Carbohydrate degrading polypeptide and uses thereof

ABSTRACT

The invention relates to a polypeptide having hemicellulase activity which comprises the amino acid sequence set out in SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71, SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or 74, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 75% sequence identity with the sequence set out in SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72 or the variant polynucleotide encodes a polypeptide that has at least 75% sequence identity with the sequence set out in SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/962,058, filed 25 Apr. 2018, which is a Continuation of U.S.application Ser. No. 14/763,263 (now U.S. Pat. No. 9,988,615), filed on24 Jul. 2015, which is a § 371 National Stage Application ofPCT/EP2014/051998, filed 3 Feb. 2014, which claims priority toEP13153824.1, filed 4 Feb. 2013, EP13156678.8, filed 26 Feb. 2013,EP13153829.0, filed 4 Feb. 2013, EP13156679.6, filed 26 Feb. 2013,EP13153831.6, filed 4 Feb. 2013, EP13156682.0, filed 26 Feb. 2013,EP13153833.2, filed 4 Feb. 2013, EP13156684.6, filed 26 Feb. 2013,EP13153834.0, filed 4 Feb. 2013, EP13156685.3, filed 26 Feb. 2013,EP13153835.7, filed 4 Feb. 2013, EP13156688.7, filed 26 Feb. 2013,EP13153836.5, filed 4 Feb. 2013, EP13156690.3, filed 26 Feb. 2013,EP13153837.3, filed 4 Feb. 2013, EP13156692.9, filed 26 Feb. 2013,EP13153839.9, filed 4 Feb. 2013, EP13156693.7, filed 26 Feb. 2013,EP13153840.7, filed 4 Feb. 2013, EP13156694.5, filed 26 Feb. 2013,EP13153841.5, filed 4 Feb. 2013, EP13156696.0, filed 26 Feb. 2013,EP13153828.2, filed 4 Feb. 2013, EP13156698.6, filed 26 Feb. 2013,EP13153825.8, filed 4 Feb. 2013, EP13156701.8, filed 26 Feb. 2013,EP13153823.3, filed 4 Feb. 2013, EP13156702.6, filed 26 Feb. 2013,EP13153821.7, filed 4 Feb. 2013, EP13156703.4, filed 26 Feb. 2013. Thedisclosure of the priority applications are incorporated in theirentirety herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2919208-341002_ST25.txt” createdon 16 Jan. 2018, and 213,889 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

The invention relates to sequences comprising genes that encodepolypeptides having lignocellulosic material degrading activity. Theinvention features the full-length coding sequence of the novel gene aswell as the amino acid sequence of the full-length functional protein,and variants and fragments of the gene or the amino acid sequence. Theinvention also relates to methods for using these proteins in industrialprocesses. Also included in the invention are cells transformed with apolynucleotide according to the invention suitable for producing theseproteins. Also the invention relates to the successful expression of thegenes that encode polypeptides having lignocellulosic material degradingactivity in a host organism such as Aspergillus niger and/or Rasamsoniaemersonii.

DESCRIPTION OF RELATED ART

Carbohydrates constitute the most abundant organic compounds on earth.However, much of this carbohydrate is sequestered in complex polymersincluding starch (the principle storage carbohydrate in seeds andgrain), and a collection of carbohydrates and lignin known aslignocellulose. The main carbohydrate components of lignocellulose arecellulose, hemicellulose, and pectins. These complex polymers are oftenreferred to collectively as lignocellulose.

Bioconversion of renewable lignocellulosic biomass to a fermentablesugar that is subsequently fermented to produce alcohol (e.g., ethanol)as an alternative to liquid fuels has attracted an intensive attentionof researchers since 1970s, when the oil crisis broke out because ofdecreasing the output of petroleum by OPEC. Ethanol has been widely usedas a 10% blend to gasoline in the USA or as a neat fuel for vehicles inBrazil in the last two decades. More recently, the use of E85, an 85%ethanol blend has been implemented especially for clean cityapplications. The importance of fuel bioethanol will increase inparallel with increases in prices for oil and the gradual depletion ofits sources. Additionally, fermentable sugars are being used to produceplastics, polymers and other bio-based products and this industry isexpected to grow substantially therefore increasing the demand forabundant low cost fermentable sugars which can be used as a feed stockin lieu of petroleum based feedstocks.

The sequestration of such large amounts of carbohydrates in plantbiomass provides a plentiful source of potential energy in the form ofsugars, both five carbon and six carbon sugars that could be utilizedfor numerous industrial and agricultural processes. However, theenormous energy potential of these carbohydrates is currentlyunder-utilized because the sugars are locked in complex polymers, andhence are not readily accessible for fermentation. Methods that generatesugars from plant biomass would provide plentiful,economically-competitive feedstocks for fermentation into chemicals,plastics, such as for instance succinic acid and (bio) fuels, includingethanol, methanol, butanol synthetic liquid fuels and biogas.

Regardless of the type of cellulosic feedstock, the cost and hydrolyticefficiency of enzymes are major factors that restrict thecommercialization of the biomass bioconversion processes. The productioncosts of microbially produced enzymes are tightly connected with aproductivity of the enzyme-producing strain and the final activity yieldin the fermentation broth.

In spite of the continued research of the last few decades to understandenzymatic lignocellulosic biomass degradation and cellulase production,it remains desirable to discover or to engineer new highly activecellulases and hemicellulases. It would also be highly desirable toconstruct highly efficient enzyme compositions capable of performingrapid and efficient biodegradation of lignocellulosic materials, inparticular such cellulases and hemicellulases that have increasedthermostability.

Such enzymes may be used to produce sugars for fermentation intochemicals, plastics, such as for instance succinic acid and (bio) fuels,including ethanol, methanol, butanol, synthetic liquid fuels and biogas,for ensiling, and also as enzyme in other industrial processes, forexample in the food or feed, textile, pulp or paper or detergentindustries and other industries.

SUMMARY

The present invention provides a polypeptide having hemicellulaseactivity or an activity according to Table 1 which comprises the aminoacid sequence set out in SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42,47, 52, 57, 62, 67 or 72 or an amino acid sequence encoded by thenucleotide sequence of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46,51, 56, 61, 66 or 71, SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49,54, 59, 64, 69 or 74, or a variant polypeptide or variant polynucleotidethereof, wherein the variant polypeptide has at least 75% sequenceidentity with the sequence set out in SEQ ID NO: 2, 7, 12, 17, 22, 27,32, 37, 42, 47, 52, 57, 62, 67 or 72 or the variant polynucleotideencodes a polypeptide that has at least 75% sequence identity with thesequence set out in SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52,57, 62, 67 or 72.

TABLE 1 Temer numbers of the proteins of the invention and theiractivity Temer number activity activity 1 Temer00088 beta-xylosidase GH32 Temer09484 beta-xylosidase GH3 3 Temer08028 alpha-galactosidase GH27 4Temer02362 alpha-galactosidase GH27 5 Temer08862 alpha-galactosidaseGH27 6 Temer04790 xyloglucanase GH12 7 Temer05249alpha-arabinofuranosidase GH51 8 Temer06848 alpha-arabinofuranosidaseGH51 9 Temer02056 alpha-arabinofuranosidase GH51 10 Temer03124endo-xylanase GH43 11 Temer09491 mannosidase/xylosidase GH31 12Temer06400 feruloyl esterase CE1 13 Temer08570 endo-xylanase GH39 14Temer08163 endo-exo-xylanase GH30 15 Temer07305 alpha-glucuronidaseGH115

The polypeptide of the invention has preferably beta-xylosidase,alpha-galactosidase, xyloglucanase, alpha-arabinofuranosidase,endo-xylanase, mannosidase/xylosidase, feruloyl esterase, xylosidase,endo-exo-xylanase or alpha-glucuronidase activity.

Furthermore the invention provides a nucleic acid sequence coding for anhemicellulase, whereby the nucleic acid sequence is selected from thegroup consisting of:

(a) a nucleic acid sequence having at least 70% identity with thenucleic acid sequence of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41,46, 51, 56, 61, 66 or 71, SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44,49, 54, 59, 64, 69 or 74 or SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70 or 75;

(b) a nucleic acid sequence hybridizing with the complement of thenucleic acid sequence of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41,46, 51, 56, 61, 66 or 71, SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44,49, 54, 59, 64, 69 or 74 or SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70 or 75;

(c) a nucleic acid sequence encoding (i) the amino acid sequence of SEQID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72, (ii)an amino acid sequence having at least 70% identity with the amino acidsequence of SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62,67 or 72, or (iii) an amino acid sequence that differs in 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12 amino acids from the amino acid sequence of SEQID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72; or

(d) a nucleotide sequence which is the reverse complement of anucleotide sequence as defined in (a), (b) or (c).

The invention also provides a nucleic acid construct or vectorcomprising the polynucleotide of the invention and a cell comprising apolypeptide of to invention or a nucleic acid construct or vector of theinvention.

According to an aspect of the invention the cell is a fungal cell,preferably a fungal cell selected from the group consisting of thegenera Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium,Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum,Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, andTrichoderma. According to another aspect of the invention one or moregene of the cell of the invention is deleted, knocked-out or disruptedin full or in part, wherein optionally the gene encodes for a protease.

The invention also provides a method for the preparation of apolypeptide according to the invention having hemicellulase or anactivity according to Table 1, which method comprises cultivating a cellof the invention under conditions which allow for expression of saidpolypeptide and, optionally, recovering the expressed polypeptide.Furthermore the invention provides a composition comprising: (i) apolypeptide of the invention and; (ii) a cellulase and/or an additionalhemicellulase and/or a pectinase, preferably the cellulase is a GH61,cellobiohydrolase, cellobiohydrolase I, cellobiohydrolase II,endo-β-1,4-glucanase, β-glucosidase or β-(1,3)(1,4)-glucanase and/or thehemicellulase is an endoxylanase, β-xylosidase, α-L-arabinofuranosidase,α-D-glucuronidase feruloyl esterase, coumaroyl esterase,α-galactosidase, β-galactosidase, β-mannanase or β-mannosidase.

Additionally the invention provides a method for the treatment of asubstrate comprising hemicellulose, optionally a plant material, whichmethod comprises contacting the substrate with a polypeptide of theinvention and/or a composition of the invention.

Another aspect of the invention relates to the use of a polypeptide ofthe invention and/or a composition of the invention to produce sugarfrom a lignocellulosic material.

The invention also provides:

a method for the preparation of a polypeptide having carbohydratematerial degrading or carbohydrate hydrolysing activity, which methodcomprises cultivating a cell of the invention under conditions whichallow for expression of said polypeptide and, optionally, recovering theexpressed polypeptide;

a polypeptide obtainable by such a method; and

a composition comprising: (i) a polypeptide of the invention and; (ii) acellulase and/or a hemicellulase and/or a pectinase;

The polypeptides of the invention having carbohydrate material degradingor carbohydrate hydrolysing activity may be used in industrialprocesses. Thus, the invention provides a method for the treatment of asubstrate comprising carbohydrate material which method comprisescontacting the substrate with a polypeptide or a composition of theinvention.

In particular, the invention provides a method for producing a sugar orsugars from lignocellulosic material which method comprises contactingthe lignocellulosic material with a polypeptide or a composition of theinvention.

Sugars produced in this way may be used in a fermentation process.Accordingly, the invention provides a method for producing afermentation product, which method comprises: producing a fermentablesugar using the described above; and fermenting the resultingfermentable sugar, thereby to produce a fermentation product.

A polypeptide or a composition of the invention may also be used, forexample, in the preparation of a food product, in the preparation of adetergent, in the preparation of an animal feed, in the treatment ofpulp or in the manufacture of a paper or in the preparation of a fabricor textile or in the cleaning thereof.

The invention also provides:

a processed material obtainable by contacting a plant material orlignocellulosic material with a polypeptide or a composition of theinvention;

a food or feed comprising a polypeptide or a composition of theinvention; and

a plant or a part thereof which comprises a polynucleotide, apolypeptide, a vector or a cell according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map of pGBTOP for expression of genes in A. niger. Depicted arethe gene of interest (GOI) expressed from the glucoamylase promoter(PglaA). In addition, the glucoamylase flank (3′glaA) of the expressioncassette is depicted. In this application a gene of interest is thecoding sequence of Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 as definedhereinafter.

FIG. 2 shows a schematic diagram of plasmid Te pep.bbn, which is thebasis for Temer00088, Temer09484, Temer08028, Temer02362, Temer08862,Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491,Temer06400, Temer08570, Temer08163 or Temer07305 overexpressionconstruct in R. emersonii that is targeted to the RePepA locus. Thevector comprises a 1500 bp 5′ flanking region 1.5 kb upstream of theRePepA ORF for targeting in the RePepA locus, a lox66 site, thenon-functional 5′ part of the ble coding region (5′ble) driven by the A.nidulans gpdA promoter, and a ccdB gene.

FIG. 3 shows a schematic diagram of plasmid pEBA1006 that was used inbipartite gene-targeting method in combination with the pEBA expressionvector containing Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 with thegoal to replace the RePepA ORF and approximately 1500 nucleotidesupstream of the start ATG codon by the expression cassette ofTemer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790,Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400,Temer08570, Temer08163 or Temer07305 in Rasamsonia emersonii. The vectorcomprises the 3′ part of the ble coding region, the A. nidulans trpCterminator, a lox71 site, a 2500 bp 3′ flanking region of the RePepAORF, and the backbone of pUC19 (Invitrogen, Breda, The Netherlands). TheE. coli DNA was removed by digestion with restriction enzyme NotI, priorto transformation of the R. emersonii strains.

FIG. 4 shows a schematic diagram of pEBA expression plasmid containingTemer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790,Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400,Temer08570, Temer08163 or Temer07305 that was used in bipartitegene-targeting method in combination with the pEBA1006 vector with thegoal to replace the RePepA ORF and approximately 1500 nucleotidesupstream of the start ATG codon by the expression cassette ofTemer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790,Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400,Temer08570, Temer08163 or Temer07305 in Rasamsonia emersonii. The vectorcomprises a 1500 bp 5′ flanking region 1.5 kb upstream of the RePepA ORFfor targeting in the RePepA locus, Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305expression cassette consisting of R. emersonii promoter 2, Temer00088,Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249,Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570,Temer08163 or Temer07305 coding region and the A. nidulans amdSterminator (TamdS), a lox66 site, the non-functional 5′ part of the blecoding region (5′ ble) driven by the A. nidulans gpdA promoter. The E.coli DNA was removed by digestion with restriction enzyme NotI, prior totransformation of the R. emersonii strains.

FIG. 5 Chromatogram obtained by High-performance anion exchangechromatography showing oligomer formation by Rasamsonia emersoniiTemer04790 in comparison with a commercial cellulase mix afterincubation on xyloglucan for 24 h incubation at pH 4.5 and 60° C.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

Table 2 shows codon-pair optimised coding sequence SEQ ID NO's, aminoacid sequence SEQ ID NO's, signal sequence SEQ ID NO's, genomic DNAsequence SEQ ID NO's and wild-type coding sequence SEQ ID NO's of thethe present invention

Codon-pair Wild-type optimized Amino acid Signal Genomic DNA codingTemer coding sequence sequence sequence sequence sequence number SEQ IDNO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 1 Temer00088 1 2 3 4 5 2Temer09484 6 7 8 9 10 3 Temer08028 11 12 13 14 15 4 Temer02362 16 17 1819 20 5 Temer08862 21 22 23 24 25 6 Temer04790 26 27 28 29 30 7Temer05249 31 32 33 34 35 8 Temer06848 36 37 38 39 40 9 Temer02056 41 4243 44 45 10 Temer03124 46 47 48 49 50 11 Temer09491 51 52 53 54 55 12Temer06400 56 57 58 59 60 13 Temer08570 61 62 63 64 65 14 Temer08163 6667 68 69 70 15 Temer07305 71 72 73 74 75

SEQ ID NO: 76 R. emersonii RePepA (genomic sequence including flanks)

SEQ ID NO: 77 R. emersonii RePepA (cDNA)

SEQ ID NO: 78 R. emersonii RePepA (protein)

SEQ ID NO: 79 A. nidulans gpdA promoter and 5′ part of the ble codingregion

SEQ ID NO: 80 3′ part of the ble coding region and A. nidulans TrpCterminator

SEQ ID NO: 81 R. emersonii promoter 2

SEQ ID NO: 82 A. nidulans AmdS terminator

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle. By way of example, “an element” may mean one element or morethan one element.

The present invention provides polynucleotides encoding polypeptides,e.g. enzymes which have the ability to modify, for example degrade, acarbohydrate material. A carbohydrate material is a material whichcomprises, consists of or substantially consists of one or morecarbohydrates. Enzymes are herein a subclass of polypeptides.

Substrate (also called feedstock) herein is used to refer to a substancethat comprises carbohydrate material, which may be treated with enzymesaccording to the invention, so that the carbohydrate material therein ismodified. In addition to the carbohydrate material the substrate maycontain any other component, including but not limited tonon-carbohydrate material and starch.

The present invention provides polynucleotides encoding polypeptides,e.g. enzymes which have the ability to modify, for example degrade, acarbohydrate material. A carbohydrate material is a material whichcomprises, consists of or substantially consists of one or morecarbohydrates. Enzymes are herein a subclass of polypeptides.

TEMER09484

Typically, a polypeptide of the invention encodes a polypeptide havingat least beta-xylosidase activity, tentatively called TEMER09484, havingan amino acid sequence according to SEQ ID NO: 2, or a sequence which isa variant thereof, typically functionally equivalent to the polypeptidehaving the sequence of SEQ ID NO: 2, or a sequence which is a fragmentof either thereof.

A β-xylosidase (EC 3.2.1.37) is any polypeptide which is capable ofcatalyzing the hydrolysis of 1,4-β-D-xylans, to remove successiveD-xylose residues from the non-reducing termini. Such enzymes may alsohydrolyze xylobiose. This enzyme may also be referred to as xylan1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase orxylobiase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of beta-xylosidase activity, for example oneof the other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER00088

Typically, a polypeptide of the invention encodes a polypeptide havingat least beta-xylosidase activity, tentatively called TEMER00088, havingan amino acid sequence according to SEQ ID NO: 7, or a sequence which isa variant thereof, typically functionally equivalent to the polypeptidehaving the sequence of SEQ ID NO: 7, or a sequence which is a fragmentof either thereof.

A β-xylosidase (EC 3.2.1.37) is any polypeptide which is capable ofcatalyzing the hydrolysis of 1,4-β-D-xylans, to remove successiveD-xylose residues from the non-reducing termini. Such enzymes may alsohydrolyze xylobiose. This enzyme may also be referred to as xylan1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase orxylobiase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of beta-xylosidase activity, for example oneof the other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER08028

Typically, a polypeptide of the invention encodes a polypeptide havingat least alpha-galactosidase activity, tentatively called TEMER08028,having an amino acid sequence according to SEQ ID NO: 12, or a sequencewhich is a variant thereof, typically functionally equivalent to thepolypeptide having the sequence of SEQ ID NO: 12, or a sequence which isa fragment of either thereof.

Herein, an α-galactosidase (EC 3.2.1.22; GH27) is any polypeptide whichis capable of catalyzing the hydrolysis of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of alpha-galactosidase activity, for exampleone of the other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER02362

Typically, a polypeptide of the invention encodes a polypeptide havingat least alpha-galactosidase activity, tentatively called TEMER02362,having an amino acid sequence according to SEQ ID NO: 17, or a sequencewhich is a variant thereof, typically functionally equivalent to thepolypeptide having the sequence of SEQ ID NO: 17, or a sequence which isa fragment of either thereof.

Herein, an α-galactosidase (EC 3.2.1.22; GH27) is any polypeptide whichis capable of catalyzing the hydrolysis of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of alpha-galactosidase activity, for exampleone of the other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER08862

Typically, a polypeptide of the invention encodes a polypeptide havingat least alpha-galactosidase activity, tentatively called TEMER08862,having an amino acid sequence according to SEQ ID NO: 22, or a sequencewhich is a variant thereof, typically functionally equivalent to thepolypeptide having the sequence of SEQ ID NO: 22, or a sequence which isa fragment of either thereof.

Herein, an α-galactosidase (EC 3.2.1.22; GH27) is any polypeptide whichis capable of catalyzing the hydrolysis of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of alpha-galactosidase activity, for exampleone of the other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER04790

Typically, a polypeptide of the invention encodes a polypeptide havingat least xyloglucanase activity, tentatively called TEMER04790, havingan amino acid sequence according to SEQ ID NO: 27, or a sequence whichis a variant thereof, typically functionally equivalent to thepolypeptide having the sequence of SEQ ID NO: 27, or a sequence which isa fragment of either thereof.

Herein, a xyloglucanase is an xyloglucan-specific endo-β-1,4-glucanase,which catalyzes the cleavage of xyloglucan, a backbone of 62 1→4-linkedglucose residues, most of which substituted with 1-6 linked xylose sidechains.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of xyloglucanase activity, for example one ofthe other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER05249

Typically, a polypeptide of the invention encodes a polypeptide havingat least alpha-arabinofuranosidase activity, tentatively calledTEMER05249, having an amino acid sequence according to SEQ ID NO: 32, ora sequence which is a variant thereof, typically functionally equivalentto the polypeptide having the sequence of SEQ ID NO: 32, or a sequencewhich is a fragment of either thereof.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of alpha-arabinofuranosidase activity, forexample one of the other carbohydrate degrading and/or carbohydratehydrolysing activities mentioned herein.

TEMER06848

Typically, a polypeptide of the invention encodes a polypeptide havingat least alpha-arabinofuranosidase activity, tentatively calledTEMER06848, having an amino acid sequence according to SEQ ID NO: 37, ora sequence which is a variant thereof, typically functionally equivalentto the polypeptide having the sequence of SEQ ID NO: 37, or a sequencewhich is a fragment of either thereof.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of alpha-arabinofuranosidase activity, forexample one of the other carbohydrate degrading and/or carbohydratehydrolysing activities mentioned herein.

TEMER02056

Typically, a polypeptide of the invention encodes a polypeptide havingat least alpha-arabinofuranosidase activity, tentatively calledTEMER02056, having an amino acid sequence according to SEQ ID NO: 42, ora sequence which is a variant thereof, typically functionally equivalentto the polypeptide having the sequence of SEQ ID NO: 42, or a sequencewhich is a fragment of either thereof.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of alpha-arabinofuranosidase activity, forexample one of the other carbohydrate degrading and/or carbohydratehydrolysing activities mentioned herein.

TEMER03124

Typically, a polypeptide of the invention encodes a polypeptide havingat least endo-xylanase activity, tentatively called TEMER03124, havingan amino acid sequence according to SEQ ID NO: 47, or a sequence whichis a variant thereof, typically functionally equivalent to thepolypeptide having the sequence of SEQ ID NO: 47, or a sequence which isa fragment of either thereof.

Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capableof catalyzing the endo-hydrolysis of 1,4-β-D-xylosidic linkages inxylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, aglucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyze1,4 xylosidic linkages in glucuronoarabinoxylans.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of endo-xylanase activity, for example one ofthe other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER09491

Typically, a polypeptide of the invention encodes a polypeptide havingat least mannosidase and/or xylosidase activity, tentatively calledTEMER09491, having an amino acid sequence according to SEQ ID NO: 52, ora sequence which is a variant thereof, typically functionally equivalentto the polypeptide having the sequence of SEQ ID NO: 52, or a sequencewhich is a fragment of either thereof.

Herein, a β-xylosidase (EC 3.2.1.37) is any polypeptide which is capableof catalyzing the hydrolysis of 1,4-β-D-xylans, to remove successiveD-xylose residues from the non-reducing termini. Such enzymes may alsohydrolyze xylobiose. This enzyme may also be referred to as xylan1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase orxylobiase.

Herein, a β-mannosidase (EC 3.2.1.25) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingβ-D-mannose residues in β-D-mannosides. This enzyme may also be referredto as mannanase or mannase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of mannosidase and/or xylosidase activity,for example one of the other carbohydrate degrading and/or carbohydratehydrolysing activities mentioned herein.

TEMER06400

Typically, a polypeptide of the invention encodes a polypeptide havingat least feruloyl esterase activity, tentatively called TEMER06400,having an amino acid sequence according to SEQ ID NO: 57, or a sequencewhich is a variant thereof, typically functionally equivalent to thepolypeptide having the sequence of SEQ ID NO: 57, or a sequence which isa fragment of either thereof.

Herein, a feruloyl esterase (EC 3.1.1.73; CE1) is any polypeptide whichis capable of catalyzing a reaction of the form:feruloyl-saccharide+H(2)O=ferulate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. It may typicallycatalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. p-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of feruloyl esterase activity, for exampleone of the other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER08570

Typically, a polypeptide of the invention encodes a polypeptide havingat least endo-xylanase activity, tentatively called TEMER08570, havingan amino acid sequence according to SEQ ID NO: 62, or a sequence whichis a variant thereof, typically functionally equivalent to thepolypeptide having the sequence of SEQ ID NO: 62, or a sequence which isa fragment of either thereof.

Herein, a β-xylosidase (EC 3.2.1.37; GH39) is any polypeptide which iscapable of catalyzing the hydrolysis of 1,4-β-D-xylans, to removesuccessive D-xylose residues from the non-reducing termini. Such enzymesmay also hydrolyze xylobiose. This enzyme may also be referred to asxylan 1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase,exo-1,4-β-xylosidase or xylobiase.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of xylosidase activity, for example one ofthe other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

TEMER08163

Typically, a polypeptide of the invention encodes a polypeptide havingat least endo- and/or exo-xylanase activity, tentatively calledTEMER08163, having an amino acid sequence according to SEQ ID NO: 67, ora sequence which is a variant thereof, typically functionally equivalentto the polypeptide having the sequence of SEQ ID NO: 67, or a sequencewhich is a fragment of either thereof. TEMER08163 advantageouslyproduces xylobiose as main product.

Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capableof catalyzing the endo-hydrolysis of 1,4-β-D-xylosidic linkages inxylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, aglucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyze1,4 xylosidic linkages in glucuronoarabinoxylans.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of endo- and/or exo-xylanase activity, forexample one of the other carbohydrate degrading and/or carbohydratehydrolysing activities mentioned herein.

TEMER07305

Typically, a polypeptide of the invention encodes a polypeptide havingat least alpha-glucuronidase activity, tentatively called TEMER07305,having an amino acid sequence according to SEQ ID NO: 72, or a sequencewhich is a variant thereof, typically functionally equivalent to thepolypeptide having the sequence of SEQ ID NO: 72, or a sequence which isa fragment of either thereof.

Herein, an α-D-glucuronidase (EC 3.2.1.139; GH115) is any polypeptidewhich is capable of catalyzing a reaction of the following form:alpha-D-glucuronoside+H(2)O=an alcohol+D-glucuronate. This enzyme mayalso be referred to as alpha-glucuronidase or alpha-glucosiduronase.These enzymes may also hydrolyze 4-O-methylated glucoronic acid, whichcan also be present as a substituent in xylans. Alternative is EC3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses thehydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.

A polypeptide of the invention may have one or more alternative and/oradditional carbohydrate degrading and/or carbohydrate hydrolysingactivities other than that of alpha-glucuronidase activity, for exampleone of the other carbohydrate degrading and/or carbohydrate hydrolysingactivities mentioned herein.

Carbohydrate in this context includes all saccharides, for examplepolysaccharides, oligosaccharides, disaccharides or monosaccharides.

A polypeptide according to the invention may modify a carbohydratematerial by chemically degrading or physically degrading such materialor hydrolysing the carbohydrate. Chemical modification of thecarbohydrate material may result in the degradation of such material,for example by hydrolysis, oxidation or other chemical modification suchas by the action of a lyase. Physical modification may or may not beaccompanied by chemical modification.

Suitable Carbohydrate Materials

Lignocellulolytic or lignocellulosic materials or biomass are abundantin nature and have great value as alternative energy source. Secondgeneration biofuels, also known as advanced biofuels, are fuels that canbe manufactured from various types of biomass. Biomass is a wide-rangingterm meaning any source of organic carbon that is renewed rapidly aspart of the carbon cycle. Biomass is derived from plant materials butcan also include animal materials. The composition of lignocellulosicbiomass varies, the major component is cellulose (35-50%), followed byxylan (20-35%, a type of hemicellulose) and lignin (10-25%), in additionto minor components such as proteins, oils and ash that make up theremaining fraction of lignocellulosic biomass. Lignocellulosic biomasscontains a variety of carbohydrates. The term carbohydrate is mostcommon in biochemistry, where it is a synonym of saccharide.Carbohydrates (saccharides) are divided into four chemical groupings:monosaccharides, disaccharides, oligosaccharides, and polysaccharides.In general, monosaccharides and disaccharides, which are smaller (lowermolecular weight) carbohydrates, are commonly referred to as sugars.

A non-starch carbohydrate suitable for modification by a polypeptide ofthe invention is lignocellulose. The major polysaccharides comprisingdifferent lignocellulosic residues, which may be considered as apotential renewable feedstock, are cellulose (glucans), hemicelluloses(xylans, heteroxylans and xyloglucans). In addition, some hemicellulosemay be present as glucomannans, for example in wood-derived feedstocks.The enzymatic hydrolysis of these polysaccharides to soluble sugars, forexample glucose, xylose, arabinose, galactose, fructose, mannose,rhamnose, ribose, D-galacturonic acid and other hexoses and pentosesoccurs under the action of different enzymes acting in concert.

Lignin fills the spaces in the cell wall between cellulose,hemicellulose, and pectin components, especially in xylem tracheids,vessel elements and sclereid cells. It is covalently linked tohemicellulose and, therefore, crosslinks different plantpolysaccharides, conferring mechanical strength to the cell wall and byextension the plant as a whole. Lignin is a highly hydrophobiccrosslinked aromatic polymeric material that is formed by differentmonolignol monomers, which can be methoxylated to various degrees. Thereare three monolignol monomers, methoxylated to various degrees:p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Theselignols are incorporated into lignin in the form of the phenylpropanoidsp-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), respectively.Biodegradation of lignin is a prerequisite for processing biofuel fromplant raw materials. Lignin can be degraded by applying differentpretreatment methods, or by using ligninases or lignin-modifying enzymes(LME's). The improving of lignin degradation would drive the output frombiofuel processing to better gain or better efficiency factor, forexample by improving the accessibility to the (hemi)cellulosiccomponents or by removing lignin-(hemi)cellulose linkages inoligosaccharides released by the action of (hemi)cellulases.

In addition, pectins and other pectic substances such as arabinans maymake up considerably proportion of the dry mass of typically cell wallsfrom non-woody plant tissues (about a quarter to half of dry mass may bepectins).

Cellulose is a linear polysaccharide composed of glucose residues linkedby β-1,4 bonds. The linear nature of the cellulose fibers, as well asthe stoichiometry of the β-linked glucose (relative to α) generatesstructures more prone to interstrand hydrogen bonding than the highlybranched α-linked structures of starch. Thus, cellulose polymers aregenerally less soluble, and form more tightly bound fibers than thefibers found in starch.

Hemicellulose is a complex polymer, and its composition often varieswidely from organism to organism and from one tissue type to another. Ingeneral, a main component of hemicellulose is β-1,4-linked xylose, afive carbon sugar. However, this xylose is often branched at O-3 and/orO-2 and can be substituted with linkages to arabinose, galactose,mannose, glucuronic acid, galacturonic acid or by esterification toacetic acid (and esterification of ferulic acid to arabinose).Hemicellulose can also contain glucan, which is a general term forβ-linked six carbon sugars (such as the β-(1,3)(1,4) glucans andheteroglucans mentioned previously) and additionally glucomannans (inwhich both glucose and mannose are present in the linear backbone,linked to each other by β-linkages).

The composition, nature of substitution, and degree of branching ofhemicellulose is very different in dicotyledonous plants (dicots, i.e.,plant whose seeds have two cotyledons or seed leaves such as lima beans,peanuts, almonds, peas, kidney beans) as compared to monocotyledonousplants (monocots; i.e., plants having a single cotyledon or seed leafsuch as corn, wheat, rice, grasses, barley). In dicots, hemicellulose iscomprised mainly of xyloglucans that are 1,4-β-linked glucose chainswith 1,6β-linked xylosyl side chains. In monocots, including most graincrops, the principal components of hemicellulose are heteroxylans. Theseare primarily comprised of 1,4-β-linked xylose backbone polymers with1,3-α linkages to arabinose, galactose, mannose and glucuronic acid or4-O-methyl-glucuronic acid as well as xylose modified by ester-linkedacetic acids. Also present are β glucans comprised of 1,3- and1,4-β-linked glucosyl chains. In monocots, cellulose, heteroxylans andβ-glucans may be present in roughly equal amounts, each comprising about15-25% of the dry matter of cell walls. Also, different plants maycomprise different amounts of, and different compositions of, pecticsubstances. For example, sugar beet contains about 19% pectin and about21% arabinan on a dry weight basis.

Accordingly, a composition of the invention may be tailored in view ofthe particular feedstock (also called substrate) which is to be used.That is to say, the spectrum of activities in a composition of theinvention may vary depending on the feedstock in question.

Enzyme combinations or physical treatments can be administeredconcomitantly or sequentially. The enzymes can be produced eitherexogenously in microorganisms, yeasts, fungi, bacteria or plants, thenisolated and added to the lignocellulosic feedstock. Alternatively, theenzymes are produced, but not isolated, and crude cell mass fermentationbroth, or plant material (such as corn stover), and the like are addedto the feedstock. Alternatively, the crude cell mass or enzymeproduction medium or plant material may be treated to prevent furthermicrobial growth (for example, by heating or addition of antimicrobialagents), then added to the feedstock. These crude enzyme mixtures mayinclude the organism producing the enzyme. Alternatively, the enzyme maybe produced in a fermentation that uses feedstock (such as corn stover)to provide nutrition to an organism that produces an enzyme(s). In thismanner, plants that produce the enzymes may serve as the lignocellulosicfeedstock and be added into lignocellulosic feedstock.

Enzymatic Activity

Endo-1,4-β-glucanases (EG) and exo-cellobiohydrolases (CBH) catalyze thehydrolysis of insoluble cellulose to cellooligosaccharides (cellobioseas a main product), while β-glucosidases (BGL) convert theoligosaccharides, mainly cellobiose and cellotriose to glucose.

Xylanases together with other accessory enzymes, for exampleα-L-arabinofuranosidases, feruloyl and acetylxylan esterases,glucuronidases, and β-xylosidases) catalyze the hydrolysis of part ofthe hemicelluloses.

Pectic substances include pectins, arabinans, galactans andarabinogalactans. Pectins are the most complex polysaccharides in theplant cell wall. They are built up around a core chain of α(1,4)-linkedD-galacturonic acid units interspersed to some degree with L-rhamnose.In any one cell wall there are a number of structural units that fitthis description and it has generally been considered that in a singlepectic molecule, the core chains of different structural units arecontinuous with one another.

Pectinases include, for example an endo-polygalacturonase, a pectinmethyl esterase, an endo-galactanase, a β-galactosidase, a pectin acetylesterase, an endo-pectin lyase, pectate lyase, α-rhamnosidase, anexo-galacturonase, an exo-polygalacturonate lyase, a rhamnogalacturonanhydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetylesterase, a rhamnogalacturonan galacturonohydrolase, axylogalacturonase, an α-arabinofuranosidase.

The principal types of structural unit are: galacturonan(homogalacturonan), which may be substituted with methanol on thecarboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I (RGI),in which galacturonic acid units alternate with rhamnose units carrying(1,4)-linked galactan and (1,5)-linked arabinan side-chains. Thearabinan side-chains may be attached directly to rhamnose or indirectlythrough the galactan chains; xylogalacturonan, with single xylosyl unitson O-3 of galacturonic acid (closely associated with RGI); andrhamnogalacturonan II (RGII), a particularly complex minor unitcontaining unusual sugars, for example apiose. An RGII unit may containtwo apiosyl residues which, under suitable ionic conditions, canreversibly form esters with borate.

As set out above, a polypeptide of the invention will typically have anactivity according to Table 1. However, a polypeptide of the inventionmay have one or more of the activities set out above in addition to oralternative to that activity. Also, a composition of the invention asdescribed herein may have one or more of the activities mentioned abovein addition to that provided by a polypeptide of the invention having anactivity according to Table 1.

Polynucleotide Sequence

The invention provides genomic polynucleotide sequences comprising thegene encoding the Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 as well asits coding sequence. Accordingly, the invention relates to an isolatedpolynucleotide comprising the genomic nucleotide sequence according tothe coding nucleotide sequence according to SEQ ID NO: 1, 6, 11, 16, 21,26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 or SEQ ID NO: 4, 9, 14, 19, 24,29, 34, 39, 44, 49, 54, 59, 64, 69 or 74 or SEQ ID NO: 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 and to variants, such asfunctional equivalents, of either thereof.

In particular, the invention relates to an isolated polynucleotide whichis capable of hybridizing selectively, for example under stringentconditions, preferably under highly stringent conditions, with thereverse complement of a polynucleotide comprising the sequence set outin SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71or in SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or74 or in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70 or 75.

More specifically, the invention relates to a polynucleotide comprisingor consisting essentially of a nucleotide sequence according to SEQ IDNO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 or SEQ IDNO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or 74 or SEQ IDNO: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75.

The invention also relates to an isolated polynucleotide comprising orconsisting essentially of a sequence which encodes at least onefunctional domain of a polypeptide according to SEQ ID NO: 2, 7, 12, 17,22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72 or a variant thereof, such asa functional equivalent, or a fragment of either thereof.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which may be isolated from chromosomal DNA, which includean open reading frame encoding a protein, e.g. the activity according tothe present invention.

A gene may include coding sequences, non-coding sequences, intronsand/or regulatory sequences. Moreover, the term “gene” may refer to anisolated nucleic acid molecule as defined herein.

A nucleic acid molecule of the present invention, such as a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 1, 6, 11, 16, 21,26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 or SEQ ID NO: 4, 9, 14, 19, 24,29, 34, 39, 44, 49, 54, 59, 64, 69 or 74 or SEQ ID NO: 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 or a variant thereof, suchas a functional equivalent, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Forexample, using all or a portion of the nucleic acid sequence of SEQ IDNO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 as ahybridization probe, nucleic acid molecules according to the inventioncan be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 or SEQID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or 74 or SEQID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 maybe isolated by the polymerase chain reaction (PCR) using syntheticoligonucleotide primers designed based upon the sequence informationcontained in SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56,61, 66 or 71 or in SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54,59, 64, 69 or 74 or in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70 or 75.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.

Furthermore, oligonucleotides corresponding to or hybridizable to anucleotide sequence according to the invention can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO: 1, 6,11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 or in SEQ ID NO: 4,9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or 74 or in SEQ ID NO:5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is the reversecomplement of the nucleotide sequence shown in SEQ ID NO: 1, 6, 11, 16,21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 or in SEQ ID NO: 4, 9, 14,19, 24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or 74 or in SEQ ID NO: 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 or a variant, suchas a functional equivalent, of either such nucleotide sequence.

A nucleic acid molecule which is complementary to another nucleotidesequence is one which is sufficiently complementary to the othernucleotide sequence such that it can hybridize to the other nucleotidesequence thereby forming a stable duplex.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode a polypeptide of the invention or a variant, such as afunctional equivalent thereof, for example a biologically activefragment or domain, as well as nucleic acid molecules sufficient for useas hybridization probes to identify nucleic acid molecules encoding apolypeptide of the invention and fragments of such nucleic acidmolecules suitable for use as PCR primers for the amplification ormutation of nucleic acid molecules.

A polynucleotide according to the invention may be “isolated”. In thecontext of this invention, an “isolated polynucleotide” or “isolatednucleic acid” is a DNA or RNA that is not immediately contiguous withone or both of the coding sequences with which it is immediatelycontiguous (one on the 5′ end and one on the 3′ end) in the naturallyoccurring genome of the organism from which it is derived. Thus, in oneembodiment, an isolated nucleic acid includes some or all of the 5′non-coding (e.g. promotor) sequences that are immediately contiguous tothe coding sequence. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector, into an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (e.g., a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other sequences. It also includes arecombinant DNA that is part of a hybrid gene encoding an additionalpolypeptide that is substantially free of cellular material, viralmaterial, or culture medium (when produced by recombinant DNAtechniques), or chemical precursors or other chemicals (when chemicallysynthesized). Moreover, an “isolated nucleic acid fragment” is a nucleicacid fragment that is not naturally occurring as a fragment and wouldnot be found in the natural state.

As used herein, the terms “polynucleotide” or “nucleic acid molecule”are intended to include DNA molecules (e.g., cDNA or genomic DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. The nucleic acidmay be synthesized using oligonucleotide analogs or derivatives (e.g.,inosine or phosphorothioate nucleotides). Such oligonucleotides can beused, for example, to prepare nucleic acids that have alteredbase-pairing abilities or increased resistance to nucleases.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305nucleic acid molecule, e.g., the coding strand of a Temer00088,Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249,Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570,Temer08163 or Temer07305 nucleic acid molecule. Also included within thescope of the invention are the complementary strands of the nucleic acidmolecules described herein.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer and all amino acid sequences of polypeptides encoded by DNAmolecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule.

The actual sequence can be more precisely determined by other approachesincluding manual DNA sequencing methods well known in the art. As isalso known in the art, a single insertion or deletion in a determinednucleotide sequence compared to the actual sequence will cause a frameshift in translation of the nucleotide sequence such that the predictedamino acid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

The person skilled in the art is capable of identifying such erroneouslyidentified bases and knows how to correct for such errors.

A nucleic acid molecule according to the invention may comprise only aportion or a fragment of the nucleic acid sequence shown in SEQ ID NO:1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 or in SEQ IDNO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or 74 or in SEQID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 (orof a variant of either thereof), for example a fragment which can beused as a probe or primer or a fragment encoding a portion of aTemer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790,Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400,Temer08570, Temer08163 or Temer07305 protein.

The nucleotide sequence determined from the cloning of the Temer00088,Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249,Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570,Temer08163 or Temer07305 gene and cDNA allows for the generation ofprobes and primers designed for use in identifying and/or cloning otherTemer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790,Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400,Temer08570, Temer08163 or Temer07305 family members, as well asTemer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790,Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400,Temer08570, Temer08163 or Temer07305 homologues from other species.

The probe/primer typically comprises a substantially purifiedoligonucleotide which typically comprises a region of nucleotidesequence that hybridizes preferably under highly stringent conditions toat least from about 12 to about 15, preferably from about 18 to about20, preferably from about 22 to about 25, more preferably about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65, orabout 75 or more consecutive nucleotides of a nucleotide sequence shownin SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71or in SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or74 or in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70 or 75 or of a variant, such as a functional equivalent, of eitherthereof.

Probes based on the Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 nucleotidesequences can be used to detect transcripts or genomic Temer00088,Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249,Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570,Temer08163 or Temer07305 sequences encoding the same or homologousproteins for instance in other organisms. In preferred embodiments, theprobe further comprises a label group attached thereto, e.g., the labelgroup can be a radioisotope, a fluorescent compound, an enzyme, or anenzyme cofactor. Such probes can also be used as part of a diagnostictest kit for identifying cells which express a TEMER09484 protein.

The polynucleotides herein may be synthetic polynucleotides. Thesynthetic polynucleotides may be optimized in codon use, preferablyaccording to the methods described in WO2006/077258 and/orPCT/EP2007/055943, which are herein incorporated by reference.PCT/EP2007/055943 addresses codon-pair optimization. Codon-pairoptimization is a method wherein the nucleotide sequences encoding apolypeptide have been modified with respect to their codon-usage, inparticular the codon-pairs that are used, to obtain improved expressionof the nucleotide sequence encoding the polypeptide and/or improvedproduction of the encoded polypeptide. Codon pairs are defined as a setof two subsequent triplets (codons) in a coding sequence.

The invention further relates to a nucleic acid construct comprising thepolynucleotide as described before. “Nucleic acid construct” is definedherein as a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which has beenmodified to contain segments of nucleic acid which are combined andjuxtaposed in a manner which would not otherwise exist in nature. Theterm nucleic acid construct is synonymous with the term “expressioncassette” when the nucleic acid construct contains all the controlsequences required for expression of a coding sequence. The term “codingsequence” as defined herein is a sequence, which is transcribed intomRNA and translated into a transcriptional activator of a proteasepromoter of the invention. The boundaries of the coding sequence aregenerally determined by the ATG start codon at the 5′end of the mRNA anda translation stop codon sequence terminating the open reading frame atthe 3′ end of the mRNA. A coding sequence can include, but is notlimited to, DNA, cDNA, and recombinant nucleic acid sequences.Preferably, the nucleic acid has high GC content. The GC content hereinindicates the number of G and C nucleotides in the construct, divided bythe total number of nucleotides, expressed in %. The GC content ispreferably 56% or more, 57% or more, 58% or more, 59% or more, 60% ormore, or in the range of 56-70% or the range of 58-65%. Preferably, theDNA construct comprises a promoter DNA sequence, a coding sequence inoperative association with said promoter DNA sequence and controlsequences such as:

-   -   one translational termination sequence orientated in 5′ towards        3′ direction selected from the following list of sequences:        TAAG, TAGA and TAAA, preferably TAAA, and/or    -   one translational initiator coding sequence orientated in 5′        towards 3′ direction selected from the following list of        sequences: GCTACCCCC; GCTACCTCC; GCTACCCTC; GCTACCTTC;        GCTCCCCCC; GCTCCCTCC; GCTCCCCTC; GCTCCCTTC; GCTGCCCCC;        GCTGCCTCC; GCTGCCCTC; GCTGCCTTC; GCTTCCCCC; GCTTCCTCC;        GCTTCCCTC; and GCTTCCTTC, preferably GCT TCC TTC, and/or    -   one translational initiator sequence selected from the following        list of sequences: 5′-mwChkyCAAA-3′; 5′-mwChkyCACA-3′ or        5′-mwChkyCAAG-3′, using ambiguity codes for nucleotides: m        (A/C); w (A/T); y (C/T); k (G/T); h (A/C/T), preferably        5′-CACCGTCAAA-3′ or 5′-CGCAGTCAAG-3′.

In the context of this invention, the term “translational initiatorcoding sequence” is defined as the nine nucleotides immediatelydownstream of the initiator or start codon of the open reading frame ofa DNA coding sequence. The initiator or start codon encodes for the AAmethionine. The initiator codon is typically ATG, but may also be anyfunctional start codon such as GTG.

In the context of this invention, the term “translational terminationsequence” is defined as the four nucleotides starting from thetranslational stop codon at the 3′ end of the open reading frame ornucleotide coding sequence and oriented in 5′ towards 3′ direction.

In the context of this invention, the term “translational initiatorsequence” is defined as the ten nucleotides immediately upstream of theinitiator or start codon of the open reading frame of a DNA sequencecoding for a polypeptide. The initiator or start codon encodes for theAA methionine. The initiator codon is typically ATG, but may also be anyfunctional start codon such as GTG. It is well known in the art thaturacil, U, replaces the deoxynucleotide thymine, T, in RNA.

Homology and Identity

Amino acid or nucleotide sequences are said to be homologous whenexhibiting a certain level of similarity. Two sequences being homologousindicate a common evolutionary origin. Whether two homologous sequencesare closely related or more distantly related is indicated by “percentidentity” or “percent similarity”, which is high or low respectively.Although disputed, to indicate “percent identity” or “percentsimilarity”, “level of homology” or “percent homology” are frequentlyused interchangeably.

The terms “homology”, “percent homology”, “percent identity” or “percentsimilarity” are used interchangeably herein. For the purpose of thisinvention, it is defined here that in order to determine the percentidentity of two amino acid sequences or of two nucleic acid sequences,the complete sequences are aligned for optimal comparison purposes. Inorder to optimize the alignment between the two sequences gaps may beintroduced in any of the two sequences that are compared. Such alignmentis carried out over the full length of the sequences being compared.Alternatively, the alignment may be carried out over a shorter length,for example over about 20, about 50, about 100 or more nucleicacids/based or amino acids. The identity is the percentage of identicalmatches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm. Theskilled person will be aware of the fact that several different computerprograms are available to align two sequences and determine the homologybetween two sequences (Kruskal, J. B. (1983) An overview of sequencecomparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, stringedits and macromolecules: the theory and practice of sequencecomparison, pp. 1-44 Addison Wesley). The percent identity between twoamino acid sequences can be determined using the Needleman and Wunschalgorithm for the alignment of two sequences. (Needleman, S. B. andWunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm alignsamino acid sequences as well as nucleotide sequences. TheNeedleman-Wunsch algorithm has been implemented in the computer programNEEDLE. For the purpose of this invention the NEEDLE program from theEMBOSS package was used (version 2.8.0 or higher, EMBOSS: The EuropeanMolecular Biology Open Software Suite (2000) Rice, P. Longden, I. andBleasby, A. Trends in Genetics 16, (6) pp 276-277,http://emboss.bioinformatics.nl). For protein sequences, EBLOSUM62 isused for the substitution matrix. For nucleotide sequences, EDNAFULL isused. Other matrices can be specified. The optional parameters used foralignment of amino acid sequences are a gap-open penalty of 10 and a gapextension penalty of 0.5. The skilled person will appreciate that allthese different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

Global Homology Definition

The homology or identity is the percentage of identical matches betweenthe two full sequences over the total aligned region including any gapsor extensions. The homology or identity between the two alignedsequences is calculated as follows: Number of corresponding positions inthe alignment showing an identical amino acid in both sequences dividedby the total length of the alignment including the gaps. The identitydefined as herein can be obtained from NEEDLE and is labelled in theoutput of the program as “IDENTITY”.

Longest Identity Definition

The homology or identity between the two aligned sequences is calculatedas follows: Number of corresponding positions in the alignment showingan identical amino acid in both sequences divided by the total length ofthe alignment after subtraction of the total number of gaps in thealignment. The identity defined as herein can be obtained from NEEDLE byusing the NOBRIEF option and is labelled in the output of the program as“longest-identity”. For purposes of the invention the level of identity(homology) between two sequences (amino acid or nucleotide) iscalculated according to the definition of “longest-identity” as can becarried out by using the program NEEDLE.

The protein sequences of the present invention can further be used as a“query sequence” to perform a search against sequence databases, forexample to identify other family members or related sequences. Suchsearches can be performed using the BLAST programs. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov).BLASTP is used for amino acid sequences and BLASTN for nucleotidesequences. The BLAST program uses as defaults:

Cost to open gap: default=5 for nucleotides/11 for proteins

Cost to extend gap: default=2 for nucleotides/1 for proteins

Penalty for nucleotide mismatch: default=−3

Reward for nucleotide match: default=1

Expect value: default=10

Wordsize: default=11 for nucleotides/28 for megablast/3 for proteins

Furthermore the degree of local identity (homology) between the aminoacid sequence query or nucleic acid sequence query and the retrievedhomologous sequences is determined by the BLAST program. However onlythose sequence segments are compared that give a match above a certainthreshold. Accordingly the program calculates the identity only forthese matching segments. Therefore the identity calculated in this wayis referred to as local identity.

Vectors

Another aspect of the invention pertains to vectors, including cloningand expression vectors, comprising a polynucleotide of the inventionencoding a TEMER09484 protein or a functional equivalent thereof andmethods of growing, transforming or transfecting such vectors in asuitable host cell, for example under conditions in which expression ofa polypeptide of the invention occurs. As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked.

Polynucleotides of the invention can be incorporated into a recombinantreplicable vector, for example a cloning or expression vector. Thevector may be used to replicate the nucleic acid in a compatible hostcell. Thus in a further embodiment, the invention provides a method ofmaking polynucleotides of the invention by introducing a polynucleotideof the invention into a replicable vector, introducing the vector into acompatible host cell, and growing the host cell under conditions whichbring about replication of the vector. The vector may be recovered fromthe host cell. Suitable host cells are described below.

The vector into which the expression cassette or polynucleotide of theinvention is inserted may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of the vectorwill often depend on the host cell into which it is to be introduced.

A vector according to the invention may be an autonomously replicatingvector, i.e. a vector which exists as an extra-chromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome (s) into which it has been integrated.

One type of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be ligated.Another type of vector is a viral vector, wherein additional DNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms “plasmid” and “vector” can be usedinterchangeably herein as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as cosmid, viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses) andphage vectors which serve equivalent functions.

Vectors according to the invention may be used in vitro, for example forthe production of RNA or used to transfect or transform a host cell.

A vector of the invention may comprise two or more, for example three,four or five, polynucleotides of the invention, for example foroverexpression.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorincludes one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed.

Within a vector, such as an expression vector, “operably linked” isintended to mean that the nucleotide sequence of interest is linked tothe regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell), i.e. the term “operably linked” refers to a juxtaposition whereinthe components described are in a relationship permitting them tofunction in their intended manner. A regulatory sequence such as apromoter, enhancer or other expression regulation signal “operablylinked” to a coding sequence is positioned in such a way that expressionof the coding sequence is achieved under condition compatible with thecontrol sequences or the sequences are arranged so that they function inconcert for their intended purpose, for example transcription initiatesat a promoter and proceeds through the DNA sequence encoding thepolypeptide.

A vector or expression construct for a given host cell may thus comprisethe following elements operably linked to each other in a consecutiveorder from the 5′-end to 3′-end relative to the coding strand of thesequence encoding the polypeptide of the first invention: (1) a promotersequence capable of directing transcription of the nucleotide sequenceencoding the polypeptide in the given host cell; (2) optionally, asignal sequence capable of directing secretion of the polypeptide fromthe given host cell into a culture medium; (3) a DNA sequence of theinvention encoding a mature and preferably active form of a polypeptidehaving cellobiohydrolase activity; and preferably also (4) atranscription termination region (terminator) capable of terminatingtranscription downstream of the nucleotide sequence encoding thepolypeptide.

Downstream of the nucleotide sequence according to the invention theremay be a 3′ untranslated region containing one or more transcriptiontermination sites (e.g. a terminator). The origin of the terminator isless critical. The terminator can, for example, be native to the DNAsequence encoding the polypeptide. However, preferably a yeastterminator is used in yeast host cells and a filamentous fungalterminator is used in filamentous fungal host cells. More preferably,the terminator is endogenous to the host cell (in which the nucleotidesequence encoding the polypeptide is to be expressed). In thetranscribed region, a ribosome binding site for translation may bepresent. The coding portion of the mature transcripts expressed by theconstructs will include a translation initiating AUG at the beginningand a termination codon appropriately positioned at the end of thepolypeptide to be translated.

Enhanced expression of the polynucleotide of the invention may also beachieved by the selection of heterologous regulatory regions, e.g.promoter, secretion leader and/or terminator regions, which may serve toincrease expression and, if desired, secretion levels of the protein ofinterest from the expression host and/or to provide for the induciblecontrol of the expression of a polypeptide of the invention.

It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. The vectors, such as expression vectors, of the invention can beintroduced into host cells to thereby produce proteins or peptides,encoded by nucleic acids as described herein (e.g. TEMER09484 proteins,mutant forms of TEMER09484 proteins, fragments, variants or functionalequivalents thereof. The vectors, such as recombinant expressionvectors, of the invention can be designed for expression of TEMER09484proteins in prokaryotic or eukaryotic cells.

For example, TEMER09484 proteins can be expressed in bacterial cellssuch as E. coli, insect cells (using baculovirus expression vectors),filamentous fungi, yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Representativeexamples of appropriate hosts are described hereafter.

Appropriate culture mediums and conditions for the above-described hostcells are known in the art.

The recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

For most filamentous fungi and yeast, the vector or expression constructis preferably integrated in the genome of the host cell in order toobtain stable transformants. However, for certain yeasts also suitableepisomal vectors are available into which the expression construct canbe incorporated for stable and high level expression, examples thereofinclude vectors derived from the 2p and pKD1 plasmids of Saccharomycesand Kluyveromyces, respectively, or vectors containing an AMA sequence(e.g. AMA1 from Aspergillus). In case the expression constructs areintegrated in the host cells genome, the constructs are eitherintegrated at random loci in the genome, or at predetermined target lociusing homologous recombination, in which case the target loci preferablycomprise a highly expressed gene.

Accordingly, expression vectors useful in the present invention includechromosomal-, episomal- and virus-derived vectors e.g., vectors derivedfrom bacterial plasmids, bacteriophage, yeast episome, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such asthose derived from plasmid and bacteriophage genetic elements, such ascosmids and phagemids.

The term “control sequences” or “regulatory sequences” is defined hereinto include at least any component which may be necessary and/oradvantageous for the expression of a polypeptide. Any control sequencemay be native or foreign to the nucleic acid sequence of the inventionencoding a polypeptide. Such control sequences may include, but are notlimited to, a promoter, a leader, optimal translation initiationsequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870),a secretion signal sequence, a pro-peptide sequence, a polyadenylationsequence, a transcription terminator. At a minimum, the controlsequences typically include a promoter, and transcriptional andtranslational stop signals. As set out above, the term “operably linked”is defined herein as a configuration in which a control sequence isappropriately placed at a position relative to the coding sequence ofthe DNA sequence such that the control sequence directs the productionof a polypeptide.

The control sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the production of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence, which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence, which shows transcriptionalactivity in the cell including mutant, truncated, and hybrid promoters,and may be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the cell.

The term “promoter” is defined herein as a DNA sequence that binds RNApolymerase and directs the polymerase to the correct downstreamtranscriptional start site of a nucleic acid sequence encoding abiological compound to initiate transcription. RNA polymeraseeffectively catalyzes the assembly of messenger RNA complementary to theappropriate DNA strand of a coding region. The term “promoter” will alsobe understood to include the 5′-non-coding region (between promoter andtranslation start) for translation after transcription into mRNA,cis-acting transcription control elements such as enhancers, and othernucleotide sequences capable of interacting with transcription factors.The promoter may be any appropriate promoter sequence suitable for aeukaryotic or prokaryotic host cell, which shows transcriptionalactivity, including mutant, truncated, and hybrid promoters, and may beobtained from polynucleotides encoding extra-cellular or intracellularpolypeptides either homologous (native) or heterologous (foreign) to thecell. The promoter may be a constitutive or inducible promoter.

Preferably the promoter is an inducible promoter. More preferably thepromoter is a carbohydrate inducible promoter. Carbohydrate induciblepromoters that are preferably used are selected from a starch-induciblepromoter (i.e. a promoter inducible by starch, a monomer, a dimer, aoligomer thereof, such as for example a maltose-inducible promoter, anisomaltose-inducible promoter), a cellulose-inducible promoter (i.e. apromoter inducible by cellulose, a monomer, a dimer and/or oligomerthereof, such as for example a cellobiose-inducible promoter, asophorose-inducible promoter), a hemicellulose inducible promoter (i.e.a promoter inducible by hemicellulose, a monomer, a dimer, and/or aoligomer thereof, such as e.g. a xylan-inducible promoter, anarabionose-inducible promoter, a xylose-inducible promoter), apectin-inducible promoter (i.e. a promoter inducible by pectin, amonomer, a dimer and/or an oligomer thereof such as for example agalacturonic acid-inducible promoter, a rhamnose-inducible promoter), anarabinan-inducible promoter (i.e. a promoter inducible by arabinan, amonomer, a dimer, and/or an oligomer thereof such as for example anarabinose-inducible promoter), a glucose-inducible promoter, alactose-inducible promoter, a galactose-inducible promoter. Otherinducible promoters are copper-, oleic acid-inducible promoters.

Promoters suitable in filamentous fungi are promoters which may beselected from the group, which includes but is not limited to promotersobtained from the polynucleotides encoding A. oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, Aspergillus gpdA promoter, A.niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A.niger or A. awamori glucoamylase (glaA), A. niger or A. awamoriendoxylanase (xlnA) or beta-xylosidase (xlnD), I reeseicellobiohydrolase I (CBHI), R. miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase,Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatumDania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusariumoxysporum trypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the polynucleotides encoding A.niger neutral alpha-amylase and A. oryzae triose phosphate isomerase),and mutant, truncated, and hybrid promoters thereof. Other examples ofpromoters are the promoters described in WO2006/092396 andWO2005/100573, which are herein incorporated by reference. An even otherexample of the use of promoters is described in WO2008/098933. Preferredcarbohydrate inducible promoters which can be used in filamentous fungiare the A. oryzae TAKA amylase, A. niger neutral alpha-amylase, A. nigeracid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA),A. niger or A. awamori endoxylanase (xlnA) or beta-xylosidase (xlnD), I,Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseIV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a hybrid of the promoters from thepolynucleotides encoding A. niger neutral alpha-amylase and A. oryzaetriose phosphate isomerase) as defined above.

Examples of such promoters from Gram-positive microorganisms include,but are not limited to, gnt (gluconate operon promoter); penP fromBacillus licheniformis; glnA (glutamine synthetase); xylAB (xyloseoperon); araABD (L-arabinose operon) and Pspac promoter, a hybridSPO1/lac promoter that can be controlled by inducers such asisopropyl-β-D-thiogalactopyranoside [IPTG] ((Yansura D. G., Henner D. J.Proc Natl Acad Sci USA. 1984 81(2):439-443). Activators are alsosequence-specific DNA binding proteins that induce promoter activity.Examples of such promoters from Gram-positive microorganisms include,but are not limited to, two-component systems (PhoP-PhoR, DegU-DegS,Spo0A-Phosphorelay), LevR, Mry and GltC. (ii) Production of secondarysigma factors can be primarily responsible for the transcription fromspecific promoters. Examples from Gram-positive microorganisms include,but are not limited to, the promoters activated by sporulation specificsigma factors: σf, σE, σG and σK and general stress sigma factor, σB.The GB-mediated response is induced by energy limitation andenvironmental stresses (Hecker M, Volker U. Mol Microbiol. 1998;29(5):1129-1136.). (iii) Attenuation and antitermination also regulatestranscription. Examples from Gram-positive microorganisms include, butare not limited to, trp operon and sacB gene. (iv) Other regulatedpromoters in expression vectors are based the sacR regulatory systemconferring sucrose inducibility (Klier A F, Rapoport G. Annu RevMicrobiol. 1988; 42:65-95).

Suitable inducible promoters useful in bacteria, such as Bacilli,include: promoters from Gram-positive microorganisms such as, but arenot limited to, SP01-26, SP01-15, veg, pyc (pyruvate carboxylasepromoter), and amyE. Examples of promoters from Gram-negativemicroorganisms include, but are not limited to, tac, tet, trp-tet, lpp,lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-PR, and λ-PL.

Additional examples of promoters useful in bacterial cells, such asBacilli, include the α-amylase and SPo2 promoters as well as promotersfrom extracellular protease genes.

Another example of a suitable promoter is the promoter obtained from theE. coli lac operon. Another example is the promoter of the Streptomycescoelicolor agarase gene (dagA). Another example is the promoter of theBacillus lentus alkaline protease gene (aprH). Another example is thepromoter of the Bacillus licheniformis alkaline protease gene(subtilisin Carlsberg gene). Another example is the promoter of theBacillus subtilis levansucrase gene (sacB). Another example is thepromoter of the Bacillus subtilis alphaamylase gene (amyF). Anotherexample is the promoter of the Bacillus licheniformis alphaamylase gene(amyL). Another example is the promoter of the Bacillusstearothermophilus maltogenic amylase gene (amyM). Another example isthe promoter of the Bacillus amyloliquefaciens alpha-amylase gene(amyQ). Another example is a “consensus” promoter having the sequenceTTGACA for the “−35” region and TATAAT for the “−10” region. Anotherexample is the promoter of the Bacillus licheniformis penicillinase gene(penP). Another example are the promoters of the Bacillus subtilis xylAand xylB genes.

Preferably the promoter sequence is from a highly expressed gene.Examples of preferred highly expressed genes from which promoters may beselected and/or which are comprised in preferred predetermined targetloci for integration of expression constructs, include but are notlimited to genes encoding glycolytic enzymes such as triose-phosphateisomerases (TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH),phosphoglycerate kinases (PGK), pyruvate kinases (PYK or PKI), alcoholdehydrogenases (ADH), as well as genes encoding amylases, glucoamylases,proteases, xylanases, cellobiohydrolases, β-galactosidases, alcohol(methanol) oxidases, elongation factors and ribosomal proteins. Specificexamples of suitable highly expressed genes include e.g. the LAC4 genefrom Kluyveromyces sp., the methanol oxidase genes (AOX and MOX) fromHansenula and Pichia, respectively, the glucoamylase (glaA) genes fromA. niger and A. awamori, the A. oryzae TAKA-amylase gene, the A.nidulans gpdA gene and the T. reesei cellobiohydrolase genes.

Promoters which can be used in yeast include e.g. promoters fromglycolytic genes, such as the phosphofructokinase (PFK), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase(GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase(PGK) promoters from yeasts or filamentous fungi; more details aboutsuch promoters from yeast may be found in (WO 93/03159). Other usefulpromoters are ribosomal protein encoding gene promoters, the lactasegene promoter (LAC4), alcohol dehydrogenase promoters (ADHI, ADH4, andthe like), and the enolase promoter (ENO). Other promoters, bothconstitutive and inducible, and enhancers or upstream activatingsequences will be known to those of skill in the art. The promoters usedin the host cells of the invention may be modified, if desired, toaffect their control characteristics. Suitable promoters in this contextinclude both constitutive and inducible natural promoters as well asengineered promoters, which are well known to the person skilled in theart. Suitable promoters in eukaryotic host cells may be GAL7, GAL10, orGAL1, CYC1, HISS, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO1,TPI1, and AOX1. Other suitable promoters include PDC1, GPD1, PGK1, TEF1,and TDH3. Examples of carbohydrate inducible promoters which can be usedare GAL promoters, such as GAL1 or GAL10 promoters.

All of the above-mentioned promoters are readily available in the art.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a filamentous fungal cell toterminate transcription. The terminator sequence is operably linked tothe 3′ terminus of the nucleic acid sequence encoding the polypeptide.Any terminator, which is functional in the cell, may be used in thepresent invention.

The control sequence may also be a terminator. Preferred terminators forfilamentous fungal cells are obtained from the genes encoding A. oryzaeTAKA amylase, A. niger glucoamylase (glaA), A. nidulans anthranilatesynthase, A. niger alpha-glucosidase, trpC gene and Fusarium oxysporumtrypsin-like protease.

The control sequence may also include a suitable leader sequence, anon-translated region of a mRNA which is important for translation bythe filamentous fungal cell. The leader sequence is operably linked tothe 5′ terminus of the nucleic acid sequence encoding the polypeptide.Any leader sequence, which is functional in the cell, may be used in thepresent invention. Preferred leaders for filamentous fungal cells areobtained from the genes encoding A. oryzae TAKA amylase and A. nidulanstriose phosphate isomerase and A. niger glaA. Other preferred sequencesare isolated and/or disclosed in WO2006/077258.

Other control sequences may be isolated from the Penicillium IPNS gene,or pcbC gene, the beta tubulin gene. All the control sequences cited inWO 01/21779 are herewith incorporated by reference.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the filamentous fungalcell as a signal to add polyadenosine residues to transcribed mRNA. Anypolyadenylation sequence, which is functional in the cell, may be usedin the present invention. Preferred polyadenylation sequences forfilamentous fungal cells are obtained from the genes encoding A. oryzaeTAKA amylase, A. niger glucoamylase, A. nidulans anthranilate synthase,Fusarium oxysporum trypsin-like protease and A. niger alpha-glucosidase.

When the polypeptide according to the invention is to be secreted fromthe host cell into the cultivation medium, an appropriate signalsequence can be added to the polypeptide in order to direct the de novosynthesized polypeptide to the secretion route of the host cell. Theperson skilled in the art knows to select an appropriate signal sequencefor a specific host. The signal sequence may be native to the host cell,or may be foreign to the host cell. As an example, a signal sequencefrom a protein native to the host cell can be used. Preferably, saidnative protein is a highly secreted protein, i.e. a protein that issecreted in amounts higher than 10% of the total amount of protein beingsecreted. The signal sequences preferably used according to theinvention are for example: pmeA.

As an alternative for a signal sequence, the polypeptide of theinvention can be fused to a secreted carrier protein, or part thereof.Such chimeric construct is directed to the secretion route by means ofthe signal sequence of the carrier protein, or part thereof. Inaddition, the carrier protein will provide a stabilizing effect to thepolypeptide according to the invention and or may enhance solubility.Such carrier protein may be any protein. Preferably, a highly secretedprotein is used as a carrier protein. The carrier protein may be nativeor foreign to the polypeptide according to the invention. The carrierprotein may be native of may be foreign to the host cell. Examples ofsuch carrier proteins are glucoamylase, prepro sequence of alpha-Matingfactor, cellulose binding domain of Clostridium cellulovorans cellulosebinding protein A, glutathione S-transferase, chitin binding domain ofBacillus circulans chitinase A1, maltose binding domain encoded by themalE gene of E. coli K12, beta-galactosidase, and alkaline phosphatase.A preferred carrier protein for expression of such chimeric construct inAspergillus cells is glucoamylase. The carrier protein and polypeptideaccording to the invention may contain a specific amino acid motif tofacilitate isolation of the polypeptide; the polypeptide according tothe invention may be released by a special releasing agent. Thereleasing agent may be a proteolytic enzyme or a chemical agent. Anexample of such amino acid motif is the KEX protease cleavage site,which is well-known to the person skilled in the art.

A signal sequence can be used to facilitate secretion and isolation of aprotein or polypeptide of the invention. Signal sequences are typicallycharacterized by a core of hydrophobic amino acids, which are generallycleaved from the mature protein during secretion in one or more cleavageevents. Such signal peptides contain processing sites that allowcleavage of the signal sequence from the mature proteins as they passthrough the secretory pathway. The signal sequence directs secretion ofthe protein, such as from a eukaryotic host into which the expressionvector is transformed, and the signal sequence is subsequently orconcurrently cleaved. The protein can then be readily purified from theextracellular medium by known methods. Alternatively, the signalsequence can be linked to the protein of interest using a sequence,which facilitates purification, such as with a GST domain. Thus, forinstance, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide, which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker sequence is a hexa-histidinepeptide, such as the tag provided in a pQE vector (Qiagen, Inc.), amongothers, many of which are commercially available. As described in Gentzet al, Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The HA tag is another peptide useful for purification whichcorresponds to an epitope derived of influenza hemaglutinin protein,which has been described by Wilson et al., Cell 37:767 (1984), forinstance.

Preferably, a TEMER09484 fusion protein of the invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers, which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A TEMER09484-encoding nucleic acid can be cloned into suchan expression vector such that the fusion moiety is linked in-frame tothe TEMER09484 protein.

(Over)expression

In a preferred embodiment, the polynucleotides of the present inventionas described herein may be over-expressed in a microbial strain of theinvention compared to the parent microbial strain in which said gene isnot over-expressed. Over-expression of a polynucleotide sequence isdefined herein as the expression of the said sequence gene which resultsin an activity of the enzyme encoded by the said sequence in a microbialstrain being at least about 1.5-fold the activity of the enzyme in theparent microbial; preferably the activity of said enzyme is at leastabout 2-fold, more preferably at least about 3-fold, more preferably atleast about 4-fold, more preferably at least about 5-fold, even morepreferably at least about 10-fold and most preferably at least about20-fold the activity of the enzyme in the parent microbial.

The vector may further include sequences flanking the polynucleotidegiving rise to RNA which comprise sequences homologous to eukaryoticgenomic sequences or viral genomic sequences. This will allow theintroduction of the polynucleotides of the invention into the genome ofa host cell.

An integrative cloning vector may integrate at random or at apredetermined target locus in the chromosome(s) of the host cell intowhich it is to be integrated. In a preferred embodiment of theinvention, an integrative cloning vector may comprise a DNA fragmentwhich is homologous to a DNA sequence in a predetermined target locus inthe genome of host cell for targeting the integration of the cloningvector to this predetermined locus. In order to promote targetedintegration, the cloning vector may be preferably linearized prior totransformation of the host cell. Linearization may preferably beperformed such that at least one but preferably either end of thecloning vector is flanked by sequences homologous to the target locus.The length of the homologous sequences flanking the target locus ispreferably at least about 0.1 kb, such as about at least 0.2 kb, morepreferably at least about 0.5 kb, even more preferably at least about 1kb, most preferably at least about 2 kb. Preferably, the parent hoststrains may be modified for improved frequency of targeted DNAintegration as described in WO05/095624 and/or WO2007/115886.

The deletion example provided in the present invention, uses thepromoter of the gene as 5′-flank and the gene as the 3′-flank to inserta selection marker between the promoter and gene, thereby disturbing(i.e. functionally inactivating) gene transcription. The gene sequencesgiven above can be used to make similar functionally inactivated genes.The genes may be split in two, yielding a 5′-flank and a 3′-flank, butthe gene may also be used to clone a larger piece of genomic DNAcontaining the promoter and terminator regions of the gene, which thancan function as 5′-flank and a 3′-flanks.

The vector system may be a single vector, such as a single plasmid, ortwo or more vectors, such as two or more plasmids, which togethercontain the total DNA to be introduced into the genome of the host cell.

The vector may contain a polynucleotide of the invention oriented in anantisense direction to provide for the production of antisense RNA.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,transduction, infection, lipofection, cationic lipid-mediatedtransfection or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2^(nd), ed. Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),Davis et al., Basic Methods in Molecular Biology (1986) and otherlaboratory manuals.

The person skilled in the art knows how to transform cells with the oneor more expression cassettes and the selectable marker. For example, theskilled person may use one or more expression vectors, wherein the oneor more cloning vectors comprise the expression cassettes and theselectable marker.

Transformation of the mutant microbial host cell may be conducted by anysuitable known methods, including e.g. electroporation methods, particlebombardment or microprojectile bombardment, protoplast methods andAgrobacterium mediated transformation (AMT). Preferably the protoplastmethod is used. Procedures for transformation are described by J. R. S.Fincham, Transformation in fungi. 1989, Microbiological reviews. 53,148-170.

Transformation may involve a process consisting of protoplast formation,transformation of the protoplasts, and regeneration of the cell wall ina manner known per se. Suitable procedures for transformation ofAspergillus cells are described in EP 238 023 and Yelton et al., 1984,Proceedings of the National Academy of Sciences USA 81:1470-1474.Suitable procedures for transformation of Aspergillus and otherfilamentous fungal host cells using Agrobacterium tumefaciens aredescribed in e.g. De Groot et al., Agrobacterium tumefaciens-mediatedtransformation of filamentous fungi. Nat Biotechnol. 1998, 16:839-842.Erratum in: Nat Biotechnol 1998 16:1074. A suitable method oftransforming Fusarium species is described by Malardier et al., 1989,Gene 78:147156 or in WO 96/00787. Other methods can be applied such as amethod using biolistic transformation as described in: Christiansen etal., Biolistic transformation of the obligate plant pathogenic fungus,Erysiphe graminis f.sp. hordei. 1995, Curr Genet. 29:100-102. Yeast maybe transformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

In order to enhance the amount of copies of the polynucleotide codingfor the compound of interest or coding for a compound involved in theproduction by the cell of the compound of interest (the gene) in themutated microbial host cell, multiple transformations of the host cellmay be required. In this way, the ratios of the different enzymesproduced by the host cell may be influenced. Also, an expression vectormay comprise multiple expression cassettes to increase the amount ofcopies of the polynucleotide(s) to be transformed.

Another way could be to choose different control sequences for thedifferent polynucleotides, which—depending on the choice—may cause ahigher or a lower production of the desired polypeptide(s).

The cells transformed with the selectable marker can be selected basedon the presence of the selectable marker. In case of transformation of(Aspergillus) cells, usually when the cell is transformed with allnucleic acid material at the same time, when the selectable marker ispresent also the polynucleotide(s) encoding the desired polypeptide(s)are present.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include, but are not limited to, thosewhich confer resistance to drugs or which complement a defect in thehost cell. They include e.g. versatile marker genes that can be used fortransformation of most filamentous fungi and yeasts such as acetamidasegenes or cDNAs (the amdS, niaD, facA genes or cDNAs from A. nidulans, A.oryzae or A. niger), or genes providing resistance to antibiotics likeG418, hygromycin, bleomycin, kanamycin, methotrexate, phleomycinorbenomyl resistance (benA). Alternatively, specific selection markerscan be used such as auxotrophic markers which require correspondingmutant host strains: e.g. URA3 (from S. cerevisiae or analogous genesfrom other yeasts), pyrG or pyrA (from A. nidulans or A. niger), argB(from A. nidulans or A. niger) or trpC. In a preferred embodiment theselection marker is deleted from the transformed host cell afterintroduction of the expression construct so as to obtain transformedhost cells capable of producing the polypeptide which are free ofselection marker genes.

Other markers include ATP synthetase, subunit 9 (oliC),orotidine-5′-phosphate decarboxylase (pvrA), the bacterial G418resistance gene (this may also be used in yeast, but not in fungi), theampicillin resistance gene (E. coli), the neomycin resistance gene(Bacillus) and the E. coli uidA gene, coding for β-glucuronidase (GUS).Vectors may be used in vitro, for example for the production of RNA orused to transfect or transform a host cell.

Expression of proteins in prokaryotes is often carried out in E. coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, e.g. to theamino terminus of the recombinant protein. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

As indicated, the expression vectors will preferably contain selectablemarkers. Such markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture and tetracyline or ampicillinresistance for culturing in E. coli and other bacteria.

Vectors preferred for use in bacteria are for example disclosed inWO-A1-2004/074468, which are hereby enclosed by reference. Othersuitable vectors will be readily apparent to the skilled artisan.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signal may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The TEMER09484 polypeptide may be expressed in a modified form, such asa fusion protein, and may include not only secretion signals but alsoadditional heterologous functional regions. Thus, for instance, a regionof additional amino acids, particularly charged amino acids, may beadded to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification

The invention provides an isolated polypeptide having the amino acidsequence according to SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47,52, 57, 62, 67 or 72, and an amino acid sequence obtainable byexpressing the polynucleotide of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31,36, 41, 46, 51, 56, 61, 66 or 71 or SEQ ID NO: 4, 9, 14, 19, 24, 29, 34,39, 44, 49, 54, 59, 64, 69 or 74 or SEQ ID NO: 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70 or 75 in an appropriate host. Also, apeptide or polypeptide comprising a variant of the above polypeptides,such as a functional equivalent, is comprised within the presentinvention. The above polypeptides are collectively comprised in the term“polypeptides according to the invention”

The term “variant peptide” or “variant polypeptide” is defined herein asa peptide or polypeptide, respectively, comprising one or morealterations, such as substitutions, insertions, deletions and/ortruncations of one or more specific amino acid residues at one or morespecific positions in the peptide or polypeptide, respectively.Accordingly, a variant signal peptide is a signal peptide comprising oneor more alterations, such as substitutions, insertions, deletions and/ortruncations of one or more specific amino acid residues at one or morespecific positions in the signal peptide.

The term “polynucleotide” is identical to the term “nucleic acidmolecule” and can herein be read interchangeably. The term refers to apolynucleotide molecule, which is a ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) molecule, either single stranded or doublestranded. A polynucleotide may either be present in isolated form, or becomprised in recombinant nucleic acid molecules or vectors, or becomprised in a host cell.

The term “variant polynucleotide” is defined herein as a polynucleotidecomprising one or more alterations, such as substitutions, insertions,deletions and/or truncations of one or more nucleotides at one or morespecific positions in the polynucleotide.

The terms “peptide” and “oligopeptide” are considered synonymous (as iscommonly recognized) and each term can be used interchangeably, as thecontext requires, to indicate a chain of at least two amino acidscoupled by peptidyl linkages. The word “polypeptide” is used herein forchains containing more than seven amino acid residues. All oligopeptideand polypeptide formulas or sequences herein are written from left toright and in the direction from amino terminus to carboxy terminus. Theone-letter code of amino acids used herein is commonly known in the artand can be found in Sambrook, et al. (Molecular Cloning: A LaboratoryManual, 2^(nd), ed. Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989)

By “isolated” polypeptide or protein is intended a polypeptide orprotein removed from its native environment. For example, recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention as are native orrecombinant polypeptides which have been substantially purified by anysuitable technique such as, for example, the single-step purificationmethod disclosed in Smith and Johnson, Gene 67:31-40 (1988).

The Temer00088, Temer09484, Temer08028, Temer02362, Temer08862,Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491,Temer06400, Temer08570, Temer08163 or Temer07305 protein according tothe invention can be recovered and purified from recombinant cellcultures by methods known in the art. Most preferably, high performanceliquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

The invention also features biologically active fragments of thepolypeptides according to the invention.

Biologically active fragments of a polypeptide of the invention includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the Temer00088, Temer09484,Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848,Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163or Temer07305 protein (e.g., the amino acid sequence of SEQ ID NO: 2, 7,12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72), which includefewer amino acids than the full length protein but which exhibit atleast one biological activity of the corresponding full-length protein.Typically, biologically active fragments comprise a domain or motif withat least one activity of the Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305protein.

A biologically active fragment of a protein of the invention can be apolypeptide which is, for example, about 10, about 25, about 50, about100 or more amino acids in length or at least about 100 amino acids, atleast 150, 200, 250, 300, 350, 400 amino acids in length, or of a lengthup the total number of amino acids of polypeptide of the invention.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the biological activities of the nativeform of a polypeptide of the invention. The invention also featuresnucleic acid fragments which encode the above biologically activefragments of the Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 protein.

Proteins

In another aspect of the invention, improved Temer00088, Temer09484,Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848,Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163or Temer07305 proteins are provided. Improved Temer00088, Temer09484,Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848,Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163or Temer07305 proteins are proteins wherein at least one biologicalactivity is improved. Such proteins may be obtained by randomlyintroducing mutations along all or part of the Temer00088, Temer09484,Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848,Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163or Temer07305 coding sequence, such as by saturation mutagenesis, andthe resulting mutants can be expressed recombinantly and screened forbiological activity. For instance, the art provides for standard assaysfor measuring the enzymatic activity of the protein of the invention andthus improved proteins may easily be selected.

Improved variants of the amino acid sequences of the present inventionleading to an improved cellobiohydrolase function may be obtained by thecorresponding genes of the present invention. Among such modificationsare included:

-   -   1. Error prone PCR to introduce random mutations, followed by a        screening of obtained variants and isolating of variants with        improved kinetic properties    -   2. Family shuffling of related variants of the genes encoding        the cellobiohydrolase, followed by a screening of obtained        variants and isolating of variants with improved kinetic        properties

Variants of the genes of the present invention leading to an increasedlevel of mRNA and/or protein, resulting in more an activity according toTable 1 may be obtained by the polynucleotide sequences of said genes.Among such modifications are included:

-   -   1. Improving the codon usage in such a way that the codons are        (optimally) adapted to the parent microbial host.    -   2. Improving the codon pair usage in such a way that the codons        are (optimally)adapted to the parent microbial host    -   3. Addition of stabilizing sequences to the genomic information        encoding the cellobiohydrolase resulting in mRNA molecules with        an increased half life

Preferred methods to isolate variants with improved catalytic propertiesor increased levels of mRNA or protein are described in WO03/010183 andWO03/01311. Preferred methods to optimize the codon usage in parentmicrobial strains are described in PCT/EP2007/05594. Preferred methodsfor the addition of stabilizing elements to the genes encoding thecellobiohydrolase of the invention are described in WO2005/059149.

In a preferred embodiment the protein of the invention has an amino acidsequence according to SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47,52, 57, 62, 67 or 72. In another embodiment, the polypeptide of theinvention is substantially homologous to the amino acid sequenceaccording to SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57,62, 67 or 72 and retains at least one biological activity of apolypeptide according to SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42,47, 52, 57, 62, 67 or 72, yet differs in amino acid sequence due tonatural variation or mutagenesis as described.

In a further preferred embodiment, the protein of the invention has anamino acid sequence encoded by an isolated nucleic acid fragment capableof hybridizing to a nucleic acid according to SEQ ID NO: 1, 6, 11, 16,21, 26, 31, 36, 41, 46, 51, 56, 61, 66 or 71 or SEQ ID NO: 4, 9, 14, 19,24, 29, 34, 39, 44, 49, 54, 59, 64, 69 or 74 or SEQ ID NO: 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75, preferably underhighly stringent hybridization conditions.

Accordingly, the Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 protein orthe protein of the invention is preferably a protein which comprises anamino acid sequence at least 50%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, 92%, 93%, 94%, 95%, 96%, 95%, 96%, 97%, 98%, 97%, 98%, 99%, 99.8%,99.9% or more homologous to the amino acid sequence shown in SEQ ID NO:2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72 and,typically, retains at least one functional activity of the polypeptideaccording to SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57,62, 67 or 72.

According to one aspect of the invention the polypeptide of theinvention may comprise the amino acid sequence set out in SEQ ID NO: 2,7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72 or an amino acidsequence that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 aminoacids from the amino acid sequence set out in SEQ ID NO: 2, 7, 12, 17,22, 27, 32, 37, 42, 47, 52, 57, 62, 67 or 72 and whereby the polypeptidestill has the activity or function of the polypeptide of the invention.The skilled person will appreciate that these minor amino acid changesin the polypeptide of the invention may be present (for examplenaturally occurring mutations) or made (for example using r-DNAtechnology) without loss of the protein function or activity. In casethese mutations are present in a binding domain, active site, or otherfunctional domain of the polypeptide a property of the polypeptide maychange (for example its thermostability) but the polypeptide may keepits hemicellulase activity. In case a mutation is present which is notclose to the active site, binding domain, or other functional domain,less effect may be expected.

Functional equivalents of a protein according to the invention can alsobe identified e.g. by screening combinatorial libraries of mutants, e.g.truncation mutants, of the protein of the invention for an activityaccording to Table 1. In one embodiment, a variegated library ofvariants is generated by combinatorial mutagenesis at the nucleic acidlevel. A variegated library of variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential protein sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g. for phage display). There are a variety ofmethods that can be used to produce libraries of potential variants ofthe polypeptides of the invention from a degenerate oligonucleotidesequence. Methods for synthesizing degenerate oligonucleotides are knownin the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al.(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide of the invention can be used to generate a variegatedpopulation of polypeptides for screening a subsequent selection ofvariants. For example, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of the codingsequence of interest with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with 51 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the protein ofinterest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations of truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In addition to the Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 genesequence shown in SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51,56, 61, 66 or 71 or in SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49,54, 59, 64, 69 or 74 or in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70 or 75, it will be apparent for the person skilled inthe art that DNA sequence polymorphisms may exist within a givenpopulation, which may lead to changes in the amino acid sequence of theTemer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790,Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400,Temer08570, Temer08163 or Temer07305 protein. Such genetic polymorphismsmay exist in cells from different populations or within a population dueto natural allelic variation. Allelic variants may also includefunctional equivalents.

Fragments of a polynucleotide according to the invention may alsocomprise polynucleotides not encoding functional polypeptides. Suchpolynucleotides may function as probes or primers for a PCR reaction.

Nucleic acids according to the invention irrespective of whether theyencode functional or non-functional polypeptides can be used ashybridization probes or polymerase chain reaction (PCR) primers. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having a Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 activityinclude, inter alia, (1) isolating the gene encoding the Temer00088,Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249,Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570,Temer08163 or Temer07305 protein, or allelic variants thereof from acDNA library e.g. from suitable microorganisms; (2) in situhybridization (e.g. FISH) to metaphase chromosomal spreads to provideprecise chromosomal location of the Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305gene as described in Verma et al., Human Chromosomes: a Manual of BasicTechniques, Pergamon Press, New York (1988); (3) Northern blot analysisfor detecting expression of Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305mRNA in specific tissues and/or cells and 4) probes and primers that canbe used as a diagnostic tool to analyse the presence of a nucleic acidhybridizable to the Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 probe in agiven biological (e.g. tissue) sample.

Also encompassed by the invention is a method of obtaining a functionalequivalent of a Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 gene. Sucha method entails obtaining a labelled probe that includes an isolatednucleic acid which encodes all or a portion of the protein sequenceaccording to SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57,62, 67 or 72 or a variant thereof; screening a nucleic acid fragmentlibrary with the labelled probe under conditions that allowhybridization of the probe to nucleic acid fragments in the library,thereby forming nucleic acid duplexes, and preparing a full-length genesequence from the nucleic acid fragments in any labelled duplex toobtain a gene related to the Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305gene.

In one embodiment, a Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 nucleicacid of the invention is at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous toa nucleic acid sequence shown in SEQ ID NO: 1, 6, 11, 16, 21, 26, 31,36, 41, 46, 51, 56, 61, 66 or 71 or in SEQ ID NO: 4, 9, 14, 19, 24, 29,34, 39, 44, 49, 54, 59, 64, 69 or 74 or in SEQ ID NO: 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 or the complement thereof.

Provided also are host cells comprising a polynucleotide or vector ofthe invention. The polynucleotide may be heterologous to the genome ofthe host cell. The term “heterologous”, usually with respect to the hostcell, means that the polynucleotide does not naturally occur in thegenome of the host cell or that the polypeptide is not naturallyproduced by that cell.

In another embodiment, the invention features cells, e.g., transformedhost cells or recombinant host cells that contain a nucleic acidencompassed by the invention. A “transformed cell” or “recombinant cell”is a cell into which (or into an ancestor of which) has been introduced,by means of recombinant DNA techniques, a nucleic acid according to theinvention. Both prokaryotic and eukaryotic cells are included, e.g.,bacteria, fungi, yeast, and the like, especially preferred are cellsfrom filamentous fungi, such as Aspergillus niger.

A host cell can be chosen that modulates the expression of the insertedsequences, or modifies and processes the gene product in a specific,desired fashion. Such modifications (e.g., glycosylation) and processing(e.g., cleavage) of protein products may facilitate optimal functioningof the protein.

Various host cells have characteristic and specific mechanisms forpost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems familiar to those ofskill in the art of molecular biology and/or microbiology can be chosento ensure the desired and correct modification and processing of theforeign protein expressed. To this end, eukaryotic host cells thatpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product canbe used. Such host cells are well known in the art.

If desired, a cell as described above may be used to in the preparationof a polypeptide according to the invention. Such a method typicallycomprises cultivating a host cell (e.g. transformed or transfected withan expression vector as described above) under conditions to provide forexpression (by the vector) of a coding sequence encoding thepolypeptide, and optionally recovering the expressed polypeptide.Polynucleotides of the invention can be incorporated into a recombinantreplicable vector, e.g. an expression vector. The vector may be used toreplicate the nucleic acid in a compatible host cell. Thus in a furtherembodiment, the invention provides a method of making a polynucleotideof the invention by introducing a polynucleotide of the invention into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about thereplication of the vector. The vector may be recovered from the hostcell.

Preferably the polypeptide is produced as a secreted protein in whichcase the nucleotide sequence encoding a mature form of the polypeptidein the expression construct is operably linked to a nucleotide sequenceencoding a signal sequence. Preferably the signal sequence is native(homologous) to the nucleotide sequence encoding the polypeptide.Alternatively the signal sequence is foreign (heterologous) to thenucleotide sequence encoding the polypeptide, in which case the signalsequence is preferably endogenous to the host cell in which thenucleotide sequence according to the invention is expressed. Examples ofsuitable signal sequences for yeast host cells are the signal sequencesderived from yeast α-factor genes. Similarly, a suitable signal sequencefor filamentous fungal host cells is e.g. a signal sequence derived froma filamentous fungal amyloglucosidase (AG) gene, e.g. the A. niger glaAgene. This may be used in combination with the amyloglucosidase (alsocalled (gluco) amylase) promoter itself, as well as in combination withother promoters. Hybrid signal sequences may also be used with thecontext of the present invention.

Preferred heterologous secretion leader sequences are those originatingfrom the fungal amyloglucosidase (AG) gene (glaA-both 18 and 24 aminoacid versions e.g. from Aspergillus), the α-factor gene (yeasts e.g.Saccharomyces and Kluyveromyces) or the α-amylase gene (Bacillus).

The vectors may be transformed or transfected into a suitable host cellas described above to provide for expression of a polypeptide of theinvention. This process may comprise culturing a host cell transformedwith an expression vector as described above under conditions to providefor expression by the vector of a coding sequence encoding thepolypeptide.

Host Cells

The invention thus provides host cells transformed or transfected withor comprising a polynucleotide or vector of the invention. Preferablythe polynucleotide is carried in a vector for the replication andexpression of the polynucleotide. The cells will be chosen to becompatible with the said vector and may for example be prokaryotic (forexample bacterial), fungal, yeast or plant cells.

A heterologous host may also be chosen wherein the polypeptide of theinvention is produced in a form which is substantially free from othercellulose-degrading or hemicellulose degrading enzymes. This may beachieved by choosing a host which does not normally produce suchenzymes.

The invention encompasses processes for the production of thepolypeptide of the invention by means of recombinant expression of a DNAsequence encoding the polypeptide. For this purpose the DNA sequence ofthe invention can be used for gene amplification and/or exchange ofexpression signals, such as promoters, secretion signal sequences, inorder to allow economic production of the polypeptide in a suitablehomologous or heterologous host cell. A homologous host cell is a hostcell which is of the same species or which is a variant within the samespecies as the species from which the DNA sequence is derived.

Suitable host cells are preferably prokaryotic microorganisms such asbacteria, or more preferably eukaryotic organisms, for example fungi,such as yeasts or filamentous fungi, or plant cells. In general, yeastcells are preferred over fungal cells because they are easier tomanipulate. However, some proteins are either poorly secreted fromyeasts, or in some cases are not processed properly (e.g.hyperglycosylation in yeast). In these instances, a fungal host organismshould be selected.

The host cell may over-express the polypeptide, and techniques forengineering over-expression are well known. The host may thus have twoor more copies of the encoding polynucleotide (and the vector may thushave two or more copies accordingly).

In the context of the present invention the “parent microbial host cell”and the “mutant microbial host cell” may be any type of host cell. Thespecific embodiments of the mutant microbial host cell are hereafterdescribed. It will be clear to those skilled in the art that embodimentsapplicable to the mutant microbial host cell are as well applicable tothe parent microbial host cell unless otherwise indicated.

The mutant microbial host cell according to the present invention may bea prokaryotic cell. Preferably, the prokaryotic host cell is bacterialcell. The term “bacterial cell” includes both Gram-negative andGram-positive microorganisms. Suitable bacteria may be selected frome.g. Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter,Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium,Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter,Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus orStreptomyces. Preferably, the bacterial cell is selected from the groupconsisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B.puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans,Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobactersphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans,E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans,Sinorhizobium melioti and Rhizobium radiobacter.

According to an embodiment, the mutant microbial host cell according tothe invention is a eukaryotic host cell. Preferably, the eukaryotic cellis a mammalian, insect, plant, fungal, or algal cell. Preferredmammalian cells include e.g. Chinese hamster ovary (CHO) cells, COScells, 293 cells, PerC6 cells, and hybridomas. Preferred insect cellsinclude e.g. Sf9 and Sf21 cells and derivatives thereof. Morepreferably, the eukaryotic cell is a fungal cell, i.e. a yeast cell,such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia strain. More preferably the eukaryotichost cell is a Kluyveromyces lactis, S. cerevisiae, Hansenulapolymorpha, Yarrowia lipolytica or Pichia pastoris, or a filamentousfungal cell. Most preferably, the eukaryotic cell is a filamentousfungal cell.

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatelyaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium,Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus,Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia,Tolypocladium, and Trichoderma.

Preferred filamentous fungal cells belong to a species of an Acremonium,Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces,Rasamsonia, Thielavia, Fusarium or Trichoderma genus, and mostpreferably a species of Aspergillus niger, Acremonium alabamense,Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae,Aspergillus fumigatus, Talaromyces emersonii, Rasamsonia emersonii,Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum,Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris orPenicillium chrysogenum. A more preferred host cell belongs to the genusAspergillus or Rasamsonia, more preferably the host cell belongs to thespecies Aspergillus niger or Rasamsonia emersonii. When the host cellaccording to the invention is an Aspergillus niger host cell, the hostcell preferably is CBS 513.88, CBS124.903 or a derivative thereof.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS),Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL), and All-Russian Collection ofMicroorganisms of Russian Academy of Sciences, (abbreviation inRussian—VKM, abbreviation in English—RCM), Moscow, Russia. Usefulstrains in the context of the present invention may be Aspergillus nigerCBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC1011, CBS205.89, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P.chrysogenum CBS 455.95, P. chrysogenum Wisconsin54-1255 (ATCC28089),Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Thielaviaterrestris NRRL8126, Talaromyces emersonii CBS 124.902, Acremoniumchrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 orATCC 56765 or ATCC 26921, Aspergillus sojae ATCC11906, Myceliophthorathermophila C1, Garg 27K, VKM-F 3500 D, Chrysosporium lucknowense C1,Garg 27K, VKM-F 3500 D, ATCC44006 and derivatives thereof.

According to one embodiment of the invention, when the mutant microbialhost cell according to the invention is a filamentous fungal host cell,the mutant microbial host cell may comprise one or more modifications inits genome such that the mutant microbial host cell is deficient in theproduction of at least one product selected from glucoamylase (glaA),acid stable alpha-amylase (amyA), neutral alpha-amylase (amyBI andamyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxinand/or fumonisin, a protease transcriptional regulator prtT, PepA, aproduct encoded by the gene hdfA and/or hdfB, a non-ribosomal peptidesynthase npsE if compared to a parent host cell and measured under thesame conditions.

Therefore, when the mutant microbial host cell according to theinvention is a filamentous fungal host cell the host cell may compriseone or more modifications in its genome to result in a deficiency in theproduction of the major extracellular aspartic protease PepA. Forexample the host cell according to the invention may further comprise adisruption of the pepA gene encoding the major extracellular asparticprotease PepA.

When the mutant microbial host cell according to the invention is afilamentous fungal host cell the host cell according to the inventionmay additionally comprises one or more modifications in its genome toresult in a deficiency in the production of the product encoded by thehdf A and/or hdfB gene. For example the host cell according to theinvention may further comprise a disruption of the hdfA and/or hdfBgene. Filamentous fungal host cells which are deficient in a productencoded by the hdfA and/or hdfB gene have been described in WO2005/095624.

When the mutant microbial host cell according to the invention is afilamentous fungal host cell the host cell according to the inventionmay additionally comprise a modification in its genome which results inthe deficiency in the production of the non-ribosomal peptide synthasenpsE. Such host cells deficient in the production of non-ribosomalpeptide synthase npsE have been described in WO2012/001169 (npsE has agenomic sequence as depicted in SEQ ID NO: 35, a coding sequencedepicted in SEQ ID NO: 36, the mRNA depicted in SEQ ID NO: 37 and thenrps protein depicted in SEQ ID NO: 38 of WO2012/001169).

When the mutant microbial host cell according to the invention is afilamentous fungal host cell the host cell may additionally comprise atleast two substantially homologous DNA domains suitable for integrationof one or more copies of a polynucleotide encoding a compound ofinterest wherein at least one of the at least two substantiallyhomologous DNA domains is adapted to have enhanced integrationpreference for the polynucleotide encoding a compound of interestcompared to the substantially homologous DNA domain it originates from,and wherein the substantially homologous DNA domain where the adaptedsubstantially homologous DNA domain originates from has a geneconversion frequency that is at least 10% higher than one of the otherof the at least two substantially homologous DNA domains. These cellshave been described in WO2011/009700. Strains containing two or morecopies of these substantially homologous DNA domains are also referredhereafter as strain containing two or more amplicons. Examples of hostcells comprising such amplicons are e.g. described in van Dijck et al,2003, Regulatory Toxicology and Pharmacology 28; 27-35: On the safety ofa new generation of DSM Aspergillus niger enzyme production strains. Invan Dijck et al, an Aspergillus niger strain is described that comprises7 amplified glucoamylase gene loci, i.e. 7 amplicons. Preferred hostcells within this context are filamentous fungus host cells, preferablyA. niger host cells, comprising two or more amplicons, preferably two ormore ΔglaA amplicons (preferably comprising 3, 4, 5, 6, 7 ΔglaAamplicons) wherein the amplicon which has the highest frequency of geneconversion, has been adapted to have enhanced integration preference forthe polynucleotide encoding a compound of interest compared to theamplicon it originates from. Adaptation of the amplicon can be performedaccording to any one of the methods described in WO2011/009700 (which ishere fully incorporated by reference). An example of these host cells,described in WO2011/009700, are host cells comprising three ΔglaAamplicons being a BamHI truncated amplicon, a SalI truncated ampliconand a BglII truncated amplicon and wherein the BamHI amplicon has beenadapted to have enhanced integration preference for a polynucleotideencoding a compound of interest compared to the BamHI amplicon itoriginates from. Host cells comprising two or more amplicons wherein oneamplicon has been adapted to have enhanced integration preference for apolynucleotide encoding a compound of interest compared to the ampliconit originates from are hereafter referred as host cells comprising anadapted amplicon.

When the mutant microbial host cell according to the invention is afilamentous fungal host cell the host cell according to the inventionmay additionally comprises a modification of Sec61. A preferred SEC61modification is a modification which results in a one-way mutant ofSEC61; i.e. a mutant wherein the de novo synthesized protein can enterthe ER via SEC61, but the protein cannot leave the ER via SEC61. Suchmodifications are extensively described in WO2005/123763. Mostpreferably, the SEC 61 modification is the S376W mutation in whichSerine 376 is replaced by Tryptophan.

Host cells according to the invention include plant cells, and theinvention therefore extends to transgenic organisms, such as plants andparts thereof, which contain one or more cells of the invention. Thecells may heterologously express the polypeptide of the invention or mayheterologously contain one or more of the polynucleotides of theinvention. The transgenic (or genetically modified) plant may thereforehave inserted (e.g. stably) into its genome a sequence encoding one ormore of the polypeptides of the invention. The transformation of plantcells can be performed using known techniques, for example using a Ti ora Ri plasmid from Agrobacterium tumefaciens. The plasmid (or vector) maythus contain sequences necessary to infect a plant, and derivatives ofthe Ti and/or Ri plasmids may be employed.

Alternatively direct infection of a part of a plant, such as a leaf,root or stem can be effected. In this technique the plant to be infectedcan be wounded, for example by cutting the plant with a razor orpuncturing the plant with a needle or rubbing the plant with anabrasive. The wound is then inoculated with the Agrobacterium. The plantor plant part can then be grown on a suitable culture medium and allowedto develop into a mature plant. Regeneration of transformed cells intogenetically modified plants can be achieved by using known techniques,for example by selecting transformed shoots using an antibiotic and bysub-culturing the shoots on a medium containing the appropriatenutrients, plant hormones and the like.

The invention also includes cells that have been modified to express thecellobiohydrolase of the invention or a variant thereof. Such cellsinclude transient, or preferably stable higher eukaryotic cell lines,such as mammalian cells or insect cells, lower eukaryotic cells, such asyeast and (e.g. filamentous) fungal cells or prokaryotic cells such asbacterial cells.

It is also possible for the proteins of the invention to be transientlyexpressed in a cell line or on a membrane, such as for example in abaculovirus expression system. Such systems, which are adapted toexpress the proteins according to the invention, are also includedwithin the scope of the present invention.

According to the present invention, the production of the polypeptide ofthe invention can be effected by the culturing of microbial expressionhosts, which have been transformed with one or more polynucleotides ofthe present invention, in a conventional nutrient fermentation medium.

Polypeptide/Enzyme Production

The recombinant host cells according to the invention may be culturedusing procedures known in the art. For each combination of a promoterand a host cell, culture conditions are available which are conducive tothe expression the DNA sequence encoding the polypeptide. After reachingthe desired cell density or titer of the polypeptide the culture isstopped and the polypeptide is recovered using known procedures.

The fermentation medium can comprise a known culture medium containing acarbon source (e.g. glucose, maltose, molasses, starch, cellulose,xylan, pectin, lignocellolytic biomass hydrolysate, etc.), a nitrogensource (e.g. ammonium sulphate, ammonium nitrate, ammonium chloride,etc.), an organic nitrogen source (e.g. yeast extract, malt extract,peptone, etc.) and inorganic nutrient sources (e.g. phosphate,magnesium, potassium, zinc, iron, etc.). Optionally, an inducer (e.g.cellulose, pectin, xylan, maltose, maltodextrin or xylogalacturonan) maybe included.

The selection of the appropriate medium may be based on the choice ofexpression host and/or based on the regulatory requirements of theexpression construct. Such media are known to those skilled in the art.The medium may, if desired, contain additional components favoring thetransformed expression hosts over other potentially contaminatingmicroorganisms.

The fermentation can be performed over a period of from about 0.5 toabout 30 days. It may be a batch, continuous or fed-batch process,suitably at a temperature in the range of 0-100° C. or 0-80° C., forexample, from about 0 to about 50° C. and/or at a pH, for example, fromabout 2 to about 10. Preferred fermentation conditions are a temperaturein the range of from about 20 to about 45° C. and/or at a pH of fromabout 3 to about 9. The appropriate conditions are usually selectedbased on the choice of the expression host and the protein to beexpressed.

After fermentation, if necessary, the cells can be removed from thefermentation broth by means of centrifugation or filtration. Afterfermentation has stopped or after removal of the cells, the polypeptideof the invention may then be recovered and, if desired, purified andisolated by conventional means.

Polypeptide/Enzyme Compositions

The invention provides a composition comprising a polypeptide of theinvention and a cellulase and/or a hemicellulase and/or a pectinaseand/or ligninase or a lignin-modifying enzyme.

When the polypeptide of the invention is a cellulase, a composition ofthe invention will typically comprise a hemicellulase and/or a pectinaseand/or ligninase or a lignin-modifying enzyme in addition to thepolypeptide of the invention.

When the polypeptide of the invention is a hemicellulase, a compositionof the invention will typically comprise a cellulase and/or a pectinaseand/or ligninase or a lignin-modifying enzyme in addition to thepolypeptide of the invention.

When the polypeptide of the invention is a pectinase, a composition ofthe invention will typically comprise a cellulase and/or a hemicellulaseand/or ligninase or a lignin-modifying enzyme in addition to thepolypeptide of the invention.

When the polypeptide of the invention is a ligninase or alignin-modifying enzyme, a composition of the invention will typicallycomprise a cellulase and/or a hemicellulase and/or a pectinase inaddition to the polypeptide of the invention.

A composition of the invention may comprise one, two or three or moreclasses of cellulase, for example one, two or all of a GH61, anendo-1,4-β-glucanase (EG), an exo-cellobiohydrolase (CBH) and aβ-glucosidase (BGL).

A composition of the invention may comprise a polypeptide which has thesame enzymatic activity, for example the same type of cellulase,hemicellulase and/or pectinase activity as that provided by apolypeptide of the invention.

A composition of the invention may comprise a polypeptide which has adifferent type of cellulase activity and/or hemicellulase activityand/or pectinase activity than that provided by a polypeptide of theinvention. For example, a composition of the invention may comprise onetype of cellulase and/or hemicellulase activity and/or pectinaseactivity provided by a polypeptide of the invention and a second type ofcellulase and/or hemicellulase activity and/or pectinase activityprovided by an additional hemicellulase/pectinase.

Herein, a cellulase is any polypeptide which is capable of degrading orcellulose. A polypeptide which is capable of degrading cellulose is onewhich is capable of catalysing the process of breaking down celluloseinto smaller units, either partially, for example into cellodextrins, orcompletely into glucose monomers. A cellulase according to the inventionmay give rise to a mixed population of cellodextrins and glucosemonomers when contacted with the cellulase. Such degradation willtypically take place by way of a hydrolysis reaction.

Herein, a hemicellulase is any polypeptide which is capable of degradingor hemicellulose. That is to say, a hemicellulase may be capable ofdegrading or one or more of xylan, glucuronoxylan, arabinoxylan,glucomannan and xyloglucan. A polypeptide which is capable of degradinga hemicellulose is one which is capable of catalysing the process ofbreaking down the hemicellulose into smaller polysaccharides, eitherpartially, for example into oligosaccharides, or completely into sugarmonomers, for example hexose or pentose sugar monomers. A hemicellulaseaccording to the invention may give rise to a mixed population ofoligosaccharides and sugar monomers when contacted with thehemicellulase. Such degradation will typically take place by way of ahydrolysis reaction.

Herein, a pectinase is any polypeptide which is capable of degrading orpectin. A polypeptide which is capable of degrading pectin is one whichis capable of catalysing the process of breaking down pectin intosmaller units, either partially, for example into oligosaccharides, orcompletely into sugar monomers. A pectinase according to the inventionmay give rise to a mixed population of oligosaccharides and sugarmonomers when contacted with the pectinase. Such degradation willtypically take place by way of a hydrolysis reaction.

Herein, a ligninase or a lignin-modifying enzyme is any polypeptidewhich is capable of degrading or modifying lignin or degradationcomponents thereof. A polypeptide which is capable of degrading ormodifying lignin is one which is capable of catalysing the process ofbreaking down lignin into smaller units, either partially, for exampleinto monophenolic compounds. A ligninase or a lignin-modifying enzymeaccording to the invention may give rise to a mixed population ofphenolic compounds when contacted with the lignin. Such degradation willtypically take place by way of an oxidation reaction. Herein, aligninase or a lignin-modifying enzyme may also be any polypeptide whichis capable of degrading phenolic degradation products of lignin. Apolypeptide which is capable of degrading phenolic degradation productsof lignin is one which is capable of catalysing the process of breakingdown phenolic degradation products of lignin into even smaller units,for example by catalysing a ring opening reaction of the phenolic ring.A ligninase or a lignin-modifying enzyme according to the invention maygive rise to a mixed population of ring-opened degradation products ofphenolic compounds when contacted with the phenolic degradation productsof lignin. Such degradation will typically take place by way of anoxidation reaction. The a ligninase or a lignin-modifying enzyme mayfurther be capable of breaking linkages between cellulose orhemicellulose and the lignin or degradation products thereof. Enzymesthat can break down lignin include lignin peroxidases, manganeseperoxidases, laccases and feruloyl esterases, and other enzymesdescribed in the art known to depolymerize or otherwise break ligninpolymers. Also included are enzymes capable of hydrolyzing bonds formedbetween hemicellulosic sugars (notably arabinose) and lignin. Ligninasesinclude but are not limited to the following group of enzymes: ligninperoxidases (EC 1.11.14), manganese peroxidases (EC 1.11.1.13), laccases(EC 1.10.3.2) and feruloyl esterases (EC 3.1.1.73).

Accordingly, a composition of the invention may comprise any cellulase,for example, a GH61, a cellobiohydrolase, an endo-β-1,4-glucanase, aβ-glucosidase or a β-(1,3)(1,4)-glucanase.

GH61 (glycoside hydrolase family 61 or sometimes referred to EGIV)proteins are oxygen-dependent polysaccharide monooxygenases (PMO's)according to the latest literature. Often in literature these proteinsare mentioned to enhance the action of cellulases on lignocellulosesubstrates. GH61 was originally classified as endogluconase based onmeasurement of very weak endo-1,4-β-d-glucanase activity in one familymember. The term “GH61” as used herein, is to be understood as a familyof enzymes, which share common conserved sequence portions and foldingsto be classified in family of the well-established CAZY GHclassification system (http://www.cazy.org/GH61.html). The glycosidehydrolase family 61 is a member of the family of glycoside hydrolases EC3.2.1. GH61 is used herein as being part of the cellulases.

Herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide which iscapable of catalysing the hydrolysis of 1,4-β-D-glucosidic linkages incellulose or cellotetraose, releasing cellobiose from the non-reducingends of the chains. This enzyme may also be referred to as cellulase1,4-β-cellobiosidase, 1,4-β-cellobiohydrolase, 1,4-β-D-glucancellobiohydrolase, avicelase, exo-1,4-β-D-glucanase,exocellobiohydrolase or exoglucanase. It may be a have the EC code EC3.2.1.91.

Herein, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptide which iscapable of catalysing the endohydrolysis of 1,4-β-D-glucosidic linkagesin cellulose, lichenin or cereal β-D-glucans. Such a polypeptide mayalso be capable of hydrolyzing 1,4-linkages in β-D-glucans alsocontaining 1,3-linkages. This enzyme may also be referred to ascellulase, avicelase, β-1,4-endoglucan hydrolase, β-1,4-glucanase,carboxymethyl cellulase, celludextrinase, endo-1,4-β-D-glucanase,endo-1,4-β-D-glucanohydrolase, endo-1,4-β-glucanase or endoglucanase.The endo-glucanase may also catalyze the cleavage of xyloglucan, abackbone of β1→4-linked glucose residues, most of which substituted with1-6 linked xylose side chains, and the enzyme is then referred to as axyloglucan-specific endo-β-1,4-glucanase or a xyloglucanase.

Herein, a β-glucosidase (EC 3.2.1.21) is any polypeptide which iscapable of catalysing the hydrolysis of terminal, non-reducingβ-D-glucose residues with release of β-D-glucose. Such a polypeptide mayhave a wide specificity for β-D-glucosides and may also hydrolyze one ormore of the following: a β-D-galactoside, an α-L-arabinoside, aβ-D-xyloside or a β-D-fucoside. This enzyme may also be referred to asamygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase.

Herein a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any polypeptide whichis capable of catalyzing the hydrolysis of 1,4-β-D-glucosidic linkagesin β-D-glucans containing 1,3- and 1,4-bonds. Such a polypeptide may acton lichenin and cereal β-D-glucans, but not on β-D-glucans containingonly 1,3- or 1,4-bonds. This enzyme may also be referred to aslicheninase, 1,3-1,4-β-D-glucan 4-glucanohydrolase, β-glucanase,endo-β-1,3-1,4 glucanase, lichenase or mixed linkage β-glucanase. Analternative for this type of enzyme is EC 3.2.1.6, which is described asendo-1,3(4)-beta-glucanase. This type of enzyme hydrolyses 1,3- or1,4-linkages in beta-D-glucans when the glucose residue whose reducinggroup is involved in the linkage to be hydrolyzed is itself substitutedat C-3. Alternative names include endo-1,3-beta-glucanase, laminarinase,1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates includelaminarin, lichenin and cereal beta-D-glucans.

A composition of the invention may comprise any hemicellulase, forexample, an endo-xylanase, a β-xylosidase, a α-L-arabionofuranosidase,an α-D-glucuronidase, an cellobiohydrolase, a feruloyl esterase, acoumaroyl esterase, an α-galactosidase, a β-galactosidase, a β-mannanaseor a β-mannosidase.

Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capableof catalyzing the endo-hydrolysis of 1,4-β-D-xylosidic linkages inxylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, aglucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyze1,4 xylosidic linkages in glucuronoarabinoxylans.

Herein, a β-xylosidase (EC 3.2.1.37; GH3) is any polypeptide which iscapable of catalyzing the hydrolysis of 1,4-β-D-xylans, to removesuccessive D-xylose residues from the non-reducing termini. Such enzymesmay also hydrolyze xylobiose. This enzyme may also be referred to asxylan 1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase,exo-1,4-β-xylosidase or xylobiase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, an α-D-glucuronidase (EC 3.2.1.139) is any polypeptide which iscapable of catalyzing a reaction of the following form:alpha-D-glucuronoside+H(2)O=an alcohol+D-glucuronate. This enzyme mayalso be referred to as alpha-glucuronidase or alpha-glucosiduronase.These enzymes may also hydrolyze 4-O-methylated glucoronic acid, whichcan also be present as a substituent in xylans. Alternative is EC3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses thehydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.

Herein, an acetyl xylan esterase (EC 3.1.1.72) is any polypeptide whichis capable of catalyzing the deacetylation of xylans andxylo-oligosaccharides. Such a polypeptide may catalyze the hydrolysis ofacetyl groups from polymeric xylan, acetylated xylose, acetylatedglucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically,not from triacetylglycerol. Such a polypeptide typically does not act onacetylated mannan or pectin.

Herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:feruloyl-saccharide+H(2)O=ferulate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. It may typicallycatalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. p-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

Herein, a coumaroyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. This enzyme mayalso be referred to as trans-4-coumaroyl esterase, trans-p-coumaroylesterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzymealso falls within EC 3.1.1.73 so may also be referred to as a feruloylesterase.

Herein, an α-galactosidase (EC 3.2.1.22) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

Herein, a β-galactosidase (EC 3.2.1.23) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingβ-D-galactose residues in β-D-galactosides. Such a polypeptide may alsobe capable of hydrolyzing α-L-arabinosides. This enzyme may also bereferred to as exo-(1->4)β-D-galactanase or lactase.

Herein, a β-mannanase (EC 3.2.1.78) is any polypeptide which is capableof catalyzing the random hydrolysis of 1,4-β-D-mannosidic linkages inmannans, galactomannans and glucomannans. This enzyme may also bereferred to as mannan endo-1,4-β-mannosidase or endo-1,4-mannanase.

Herein, a β-mannosidase (EC 3.2.1.25) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingβ-D-mannose residues in β-D-mannosides. This enzyme may also be referredto as mannanase or mannase.

A composition of the invention may comprise any pectinase, for examplean endo polygalacturonase, a pectin methyl esterase, anendo-galactanase, a beta galactosidase, a pectin acetyl esterase, anendo-pectin lyase, pectate lyase, alpha rhamnosidase, anexo-galacturonase, an exo-polygalacturonate lyase, a rhamnogalacturonanhydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetylesterase, a rhamnogalacturonan galacturonohydrolase or axylogalacturonase.

Herein, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide whichis capable of catalyzing the random hydrolysis of1,4-α-D-galactosiduronic linkages in pectate and other galacturonans.This enzyme may also be referred to as polygalacturonase pectindepolymerase, pectinase, endopolygalacturonase, pectolase, pectinhydrolase, pectin polygalacturonase, poly-α-1,4-galacturonideglycanohydrolase, endogalacturonase; endo-D-galacturonase orpoly(1,4-α-D-galacturonide) glycanohydrolase.

Herein, a pectin methyl esterase (EC 3.1.1.11) is any enzyme which iscapable of catalyzing the reaction: pectin+n H₂O=n methanol+pectate. Theenzyme may also been known as pectinesterase, pectin demethoxylase,pectin methoxylase, pectin methylesterase, pectase, pectinoesterase orpectin pectylhydrolase.

Herein, an endo-galactanase (EC 3.2.1.89) is any enzyme capable ofcatalyzing the endohydrolysis of 1,4-β-D-galactosidic linkages inarabinogalactans. The enzyme may also be known as arabinogalactanendo-1,4-β-galactosidase, endo-1,4-β-galactanase, galactanase,arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.

Herein, a pectin acetyl esterase is defined herein as any enzyme whichhas an acetyl esterase activity which catalyzes the deacetylation of theacetyl groups at the hydroxyl groups of GalUA residues of pectin

Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable ofcatalyzing the eliminative cleavage of (1→4)-α-D-galacturonan methylester to give oligosaccharides with4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducingends. The enzyme may also be known as pectin lyase, pectintrans-eliminase; endo-pectin lyase, polymethylgalacturonictranseliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGLor (1→4)-6-O-methyl-α-D-galacturonan lyase.

Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzingthe eliminative cleavage of (1→4)-α-D-galacturonan to giveoligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known polygalacturonictranseliminase, pectic acid transeliminase, polygalacturonate lyase,endopectin methyltranseliminase, pectate transeliminase,endogalacturonate transeliminase, pectic acid lyase, pectic lyase,α-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase,pectin trans-eliminase, polygalacturonic acid trans-eliminase or(1→4)-α-D-galacturonan lyase.

Herein, an alpha rhamnosidase (EC 3.2.1.40) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingα-L-rhamnose residues in α-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T,α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.

Herein, exo-galacturonase (EC 3.2.1.82) is any polypeptide capable ofhydrolysis of pectic acid from the non-reducing end, releasingdigalacturonate. The enzyme may also be known asexo-poly-α-galacturonosidase, exopolygalacturonosidase orexopolygalacturanosidase.

Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable ofcatalyzing:(1,4-α-D-galacturonide)_(n)+H₂O=(1,4-α-D-galacturonide)_(n-1)+D-galacturonate.The enzyme may also be known as galacturan 1,4-α-galacturonidase,exopolygalacturonase, poly(galacturonate) hydrolase,exo-D-galacturonase, exo-D-galacturonanase, exo-poly-D-galacturonase orpoly(1,4-α-D-galacturonide) galacturonohydrolase.

Herein, exo-polygalacturonate lyase (EC 4.2.2.9) is any polypeptidecapable of catalyzing eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate, i.e. de-esterified pectin. This enzyme may be known aspectate disaccharide-lyase, pectate exo-lyase, exopectic acidtranseliminase, exo-pectate lyase, exopolygalacturonicacid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-α-D-galacturonanreducing-end-disaccharide-lyase.

Herein, rhamnogalacturonan hydrolase is any polypeptide which is capableof hydrolyzing the linkage between galactosyluronic acid andrhamnopyranosyl in an endo-fashion in strictly alternatingrhamnogalacturonan structures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

Herein, rhamnogalacturonan lyase is any polypeptide which is anypolypeptide which is capable of cleaving α-L-Rhap-(1→4)-α-D-GalpAlinkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

Herein, rhamnogalacturonan acetyl esterase is any polypeptide whichcatalyzes the deacetylation of the backbone of alternating rhamnose andgalacturonic acid residues in rhamnogalacturonan.

Herein, rhamnogalacturonan galacturonohydrolase is any polypeptide whichis capable of hydrolyzing galacturonic acid from the non-reducing end ofstrictly alternating rhamnogalacturonan structures in an exo-fashion.

Herein, xylogalacturonase is any polypeptide which acts onxylogalacturonan by cleaving the β-xylose substituted galacturonic acidbackbone in an endo-manner. This enzyme may also be known asxylogalacturonan hydrolase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide which iscapable of catalyzing endohydrolysis of 1,5-α-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be know asendo-arabinase, arabinan endo-1,5-α-L-arabinosidase,endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase; endo-arabanase or1,5-α-L-arabinan 1,5-α-L-arabinanohydrolase.

A composition of the invention will typically comprise at least onecellulase and/or at least one hemicellulase and/or at least onepectinase (one of which is a polypeptide according to the invention). Acomposition of the invention may comprise a cellobiohydrolase, anendoglucanase and/or a β-glucosidase. Such a composition may alsocomprise one or more hemicellulases and/or one or more pectinases.

One or more (for example two, three, four or all) of an amylase, aprotease, a lipase, a ligninase, a hexosyltransferase or a glucuronidasemay be present in a composition of the invention.

“Protease” includes enzymes that hydrolyze peptide bonds (peptidases),as well as enzymes that hydrolyze bonds between peptides and othermoieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4, and are suitable for use in the inventionincorporated herein by reference. Some specific types of proteasesinclude, cysteine proteases including pepsin, papain and serineproteases including chymotrypsins, carboxypeptidases andmetalloendopeptidases.

“Lipase” includes enzymes that hydrolyze lipids, fatty acids, andacylglycerides, including phospoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

“Hexosyltransferase” (2.4.1-) includes enzymes which are capable oftransferring glycosyl groups, more specifically hexosyl groups. Inaddition to transfer of a glycosyl-group from a glycosyl-containingdonor to another glycosyl-containing compound, the acceptor, the enzymescan also transfer the glycosyl-group to water as an acceptor. Thisreaction is also known as a hydrolysis reaction, instead of a transferreaction. An example of a hexosyltransferase which may be used in theinvention is a β-glucanosyltransferase. Such an enzyme may be able tocatalyze degradation of (1,3)(1,4)glucan and/or cellulose and/or acellulose degradation product.

“Glucuronidase” includes enzymes that catalyze the hydrolysis of aglucoronoside, for example β-glucuronoside to yield an alcohol. Manyglucuronidases have been characterized and may be suitable for use inthe invention, for example β-glucuronidase (EC 3.2.1.31),hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamineglucuronidase (3.2.1.56), glycyrrhizinate β-glucuronidase (3.2.1.128) orα-D-glucuronidase (EC 3.2.1.139).

A composition of the invention may comprise an expansin or expansin-likeprotein, such as a swollenin (see Salheimo et al., Eur. J. Biochem. 269,4202-4211, 2002) or a swollenin-like protein.

Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Forthe purposes of this invention, an expansin-like protein orswollenin-like protein may comprise one or both of such domains and/ormay disrupt the structure of cell walls (such as disrupting cellulosestructure), optionally without producing detectable amounts of reducingsugars.

A composition of the invention may comprise the polypeptide product of acellulose integrating protein, scaffoldin or a scaffoldin-like protein,for example CipA or CipC from Clostridium thermocellum or Clostridiumcellulolyticum respectively.

Scaffoldins and cellulose integrating proteins are multi-functionalintegrating subunits which may organize cellulolytic subunits into amulti-enzyme complex. This is accomplished by the interaction of twocomplementary classes of domain, i.e. a cohesion domain on scaffoldinand a dockerin domain on each enzymatic unit. The scaffoldin subunitalso bears a cellulose-binding module (CBM) that mediates attachment ofthe cellulosome to its substrate. A scaffoldin or cellulose integratingprotein for the purposes of this invention may comprise one or both ofsuch domains.

A composition of the invention may comprise a cellulose induced proteinor modulating protein, for example as encoded by cip1 or cip2 gene orsimilar genes from Trichoderma reesei/Hypocrea jacorina (see Foreman etal., J. Biol. Chem. 278(34), 31988-31997, 2003). The polypeptide productof these genes are bimodular proteins, which contain a cellulose bindingmodule and a domain which function or activity can not be related toknown glycosyl hydrolase families. Yet, the presence of a cellulosebinding module and the co-regulation of the expression of these geneswith cellulases components indicates previously unrecognized activitieswith potential role in biomass degradation.

A composition of the invention may be composed of a member of each ofthe classes of the polypeptides mentioned above, several members of onepolypeptide class, or any combination of these polypeptide classes.

A composition of the invention may be composed of polypeptides, forexample enzymes, from (1) commercial suppliers; (2) cloned genesexpressing polypeptides, for example enzymes; (3) complex broth (such asthat resulting from growth of a microbial strain in media, wherein thestrains secrete proteins and enzymes into the media; (4) cell lysates ofstrains grown as in (3); and/or (5) plant material expressingpolypeptides, for example enzymes. Different polypeptides, for exampleenzymes in a composition of the invention may be obtained from differentsources.

Use of the Polypeptides

The polypeptides and polypeptide compositions according to the inventionmay be used in many different applications. For instance they may beused to produce fermentable sugars. The fermentable sugars can then, aspart of a biofuel process, be converted into biogas or ethanol, butanol,isobutanol, 2 butanol or other suitable substances. Alternatively thepolypeptides and their compositions may be used as enzyme, for instancein production of food products, in detergent compositions, in the paperand pulp industry and in antibacterial formulations, in pharmaceuticalproducts such as throat lozenges, toothpastes, and mouthwash. Some ofthe uses will be illustrated in more detail below.

In the uses and methods described below, the components of thecompositions described above may be provided concomitantly (i.e. as asingle composition per se) or separately or sequentially.

The invention also relates to the use of the cellobiohydrolase accordingto the invention and compositions comprising such an enzyme inindustrial processes.

Despite the long term experience obtained with these processes, thecellobiohydrolase according to the invention may feature a number ofsignificant advantages over enzymes currently used. Depending on thespecific application, these advantages may include aspects such as lowerproduction costs, higher specificity towards the substrate, reducedantigenicity, fewer undesirable side activities, higher yields whenproduced in a suitable microorganism, more suitable pH and temperatureranges, non-inhibition by hydrophobic, lignin-derived products or lessproduct inhibition or, in the case of the food industry a better tasteor texture of a final product as well as food grade and kosher aspects.

In principle, a cellobiohydrolase or composition of the invention may beused in any process which requires the treatment of a material whichcomprises polysaccharide. Thus, a polypeptide or composition of theinvention may be used in the treatment of polysaccharide material.Herein, polysaccharide material is a material which comprises orconsists essential of one or, more typically, more than onepolysaccharide.

Typically, plants and material derived therefrom comprise significantquantities of non-starch polysaccharide material. Accordingly, apolypeptide of the invention may be used in the treatment of a plant orfungal material or a material derived therefrom.

Lignocellulose

An important component of plant non-starch polysaccharide material islignocellulose (also referred to herein as lignocellulolytic biomass).Lignocellulose is plant material that comprises cellulose andhemicellulose and lignin. The carbohydrate polymers (cellulose andhemicelluloses) are tightly bound to the lignin by hydrogen and covalentbonds. Accordingly, a polypeptide of the invention may be used in thetreatment of lignocellulolytic material. Herein, lignocellulolyticmaterial is a material which comprises or consists essential oflignocellulose. Thus, in a method of the invention for the treatment ofa non-starch polysaccharide, the non-starch polysaccharide may be alignocellulosic material/biomass.

Accordingly, the invention provides a method of treating a substratecomprising non-starch polysaccharide in which the treatment comprisesthe degradation and/or hydrolysis and/or modification of celluloseand/or hemicellulose and/or a pectic substance.

Degradation in this context indicates that the treatment results in thegeneration of hydrolysis products of cellulose and/or hemicelluloseand/or a pectic substance, i.e. saccharides of shorter length arepresent as result of the treatment than are present in a similaruntreated non-starch polysaccharide. Thus, degradation in this contextmay result in the liberation of oligosaccharides and/or sugar monomers.

All plants contain non-starch polysaccharide as do virtually allplant-derived polysaccharide materials. Accordingly, in a method of theinvention for the treatment of substrate comprising a non-starchpolysaccharide, said substrate may be provided in the form of a plant ora plant derived material or a material comprising a plant or plantderived material, for example a plant pulp, a plant extract, a foodstuffor ingredient therefore, a fabric, a textile or an item of clothing.

Lignocellulolytic biomass suitable for use in the invention includesbiomass and can include virgin biomass and/or non-virgin biomass such asagricultural biomass, commercial organics, construction and demolitiondebris, municipal solid waste, waste paper and yard waste. Common formsof biomass include trees, shrubs and grasses, wheat, wheat straw, sugarcane bagasse, corn, corn husks, corn cobs, corn kernel including fiberfrom kernels, products and by-products from milling of grains such ascorn, wheat and barley (including wet milling and dry milling) oftencalled “bran or fiber” as well as municipal solid waste, waste paper andyard waste. The biomass can also be, but is not limited to, herbaceousmaterial, agricultural residues, forestry residues, municipal solidwastes, waste paper, and pulp and paper mill residues. “Agriculturalbiomass” includes branches, bushes, canes, corn and corn husks, energycrops, forests, fruits, flowers, grains, grasses, herbaceous crops,leaves, bark, needles, logs, roots, saplings, short rotation woodycrops, shrubs, switch grasses, trees, vegetables, fruit peels, vines,sugar beet pulp, wheat middlings, oat hulls, and hard and soft woods(not including woods with deleterious materials). In addition,agricultural biomass includes organic waste materials generated fromagricultural processes including farming and forestry activities,specifically including forestry wood waste. Agricultural biomass may beany of the aforestated singularly or in any combination or mixturethereof. Further examples of suitable biomass are orchard primings,chaparral, mill waste, urban wood waste, municipal waste, logging waste,forest thinnings, short-rotation woody crops, industrial waste, wheatstraw, oat straw, rice straw, barley straw, rye straw, flax straw, soyhulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane,corn stover, corn stalks, corn cobs, corn husks, prairie grass,gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls,cellulosic animal wastes, lawn clippings, cotton, seaweed, trees,shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, cornhusks, corn hobs, corn kernel, fiber from kernels, products andby-products from wet or dry milling of grains, municipal solid waste,waste paper, yard waste, herbaceous material, agricultural residues,forestry residues, municipal solid waste, waste paper, pulp, paper millresidues, branches, bushes, canes, corn, corn husks, an energy crop,forest, a fruit, a flower, a grain, a grass, a herbaceous crop, a leaf,bark, a needle, a log, a root, a sapling, a shrub, switch grass, a tree,a vegetable, fruit peel, a vine, sugar beet pulp, wheat middlings, oathulls, hard or soft wood, organic waste material generated from anagricultural process, forestry wood waste, or a combination of any twoor more thereof.

Apart from virgin biomass or feedstocks already processed in food andfeed or paper and pulping industries, the biomass/feedstock mayadditionally be pretreated with heat, mechanical and/or chemicalmodification or any combination of such methods in order to enhanceenzymatic degradation.

Pretreatment

Before enzymatic treatment, the feedstock may optionally be pre-treatedwith heat, mechanical and/or chemical modification or any combination ofsuch methods in order to to enhance the accessibility of the substrateto enzymatic hydrolysis and/or hydrolyse the hemicellulose and/orsolubilize the hemicellulose and/or cellulose and/or lignin, in any wayknown in the art. The pretreatment may comprise exposing thelignocellulosic material to (hot) water, steam (steam explosion), anacid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding,grinding, milling or rapid depressurization, or a combination of any twoor more thereof. This chemical pretreatment is often combined withheat-pretreatment, e.g. between 150-220° C. for 1 to 30 minutes.

Presaccharifation

After the pretreatment step, a liquefaction/hydrolysis orpresaccharification step involving incubation with an enzyme or enzymemixture can be utilized. The presaccharification step can be performedat many different temperatures but it is preferred that thepresaccharification step occur at the temperature best suited to theenzyme mix being tested, or the predicted enzyme optimum of the enzymesto be tested. The temperature of the presaccharification step may rangefrom about 10° C. to about 95° C., about 20° C. to about 85° C., about30° C. to about 70° C., about 40° C. to about 60° C., about 37° C. toabout 50° C., preferably about 37° C. to about 80° C., more preferablyabout 60-70° C. even more preferably around 65° C. The pH of thepresaccharification mixture may range from about 2.0 to about 10.0, butis preferably about 3.0 to about 7.0, more preferably about 4.0 to about6.0, even more preferably about 4.0 to about 5.0. Again, the pH may beadjusted to maximize enzyme activity and may be adjusted with theaddition of the enzyme. Comparison of the results of the assay resultsfrom this test will allow one to modify the method to best suit theenzymes being tested.

The liquefaction/hydrolysis or presaccharification step reaction mayoccur from several minutes to several hours, such as from about 1 hourto about 120 hours, preferably from about 2 hours to about 48 hours,more preferably from about 2 to about 24 hours, most preferably for fromabout 2 to about 6 hours. The cellulase treatment may occur from severalminutes to several hours, such as from about 6 hours to about 120 hours,preferably about 12 hours to about 72 hours, more preferably about 24 to48 hours.

Saccharification

The invention provides a method for producing a sugar from alignocellulosic material which method comprises contacting a polypeptideof the invention to a composition of the invention with thelignocellulosic material.

Such a method allows free sugars (monomers) and/or oligosaccharides tobe generated from lignocellulosic biomass. These methods involveconverting lignocellulosic biomass to free sugars and smalloligosaccharides with a polypeptide or composition of the invention.

The process of converting a complex carbohydrate such as lignocelluloseinto sugars preferably allows conversion into fermentable sugars. Such aprocess may be referred to as “saccharification.” Accordingly, a methodof the invention may result in the liberation of one or more hexoseand/or pentose sugars, such as one or more of glucose, xylose,arabinose, galactose, galacturonic acid, glucuronic acid, mannose,rhamnose, ribose and fructose.

Accordingly, another aspect of the invention includes methods thatutilize the polypeptide of composition of the invention described abovetogether with further enzymes or physical treatments such as temperatureand pH to convert the lignocellulosic plant biomass to sugars andoligosaccharides.

While the composition has been discussed as a single mixture it isrecognized that the enzymes may be added sequentially where thetemperature, pH, and other conditions may be altered to increase theactivity of each individual enzyme. Alternatively, an optimum pH andtemperature can be determined for the enzyme mixture.

The enzymes are reacted with substrate under any appropriate conditions.For example, enzymes can be incubated at about 25° C., about 30° C.,about 35° C., about 37° C., about 40° C., about 45° C., about 50° C.,about 55° C., about 60° C., about 65° C., about 70° C., about 75° C.,about 80° C., about 85° C., about 90° C. or higher. That is, they can beincubated at a temperature of from about 20° C. to about 95° C., forexample in buffers of low to medium ionic strength and/or from low toneutral pH. By “medium ionic strength” is intended that the buffer hasan ion concentration of about 200 millimolar (mM) or less for any singleion component. The pH may range from about pH 2.5, about pH 3.0, aboutpH 3.5, about pH 4.0, about pH 4.5, about pH 5, about pH 5.5, about pH6, about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, to about pH8.5. Generally, the pH range will be from about pH 3.0 to about pH 7.For the production of ethanol an acidic medium is preferred, e.g. pH=4,whereas for the production of biogas neutral pH, e.g. pH=7 is desirable.Incubation of enzyme combinations under these conditions results inrelease or liberation of substantial amounts of the sugar from thelignocellulose. By substantial amount is intended at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or more of available sugar.

The polypeptides, such as enzymes, can be produced either exogenously inmicroorganisms, yeasts, fungi, bacteria or plants, then isolated andadded, for example, to lignocellulosic feedstock. Alternatively, theenzymes are produced, but not isolated, and crude cell mass fermentationbroth, or plant material (such as corn stover), and the like may beadded to, for example, the feedstock. Alternatively, the crude cell massor enzyme production medium or plant material may be treated to preventfurther microbial growth (for example, by heating or addition ofantimicrobial agents), then added to, for example, a feedstock. Thesecrude enzyme mixtures may include the organism producing the enzyme.Alternatively, the enzyme may be produced in a fermentation that usesfeedstock (such as corn stover) to provide nutrition to an organism thatproduces an enzyme(s). In this manner, plants that produce the enzymesmay themselves serve as a lignocellulosic feedstock and be added intolignocellulosic feedstock.

Fermentation of Sugars

The fermentable sugars can be converted to useful value-addedfermentation products, non-limiting examples of which include aminoacids, vitamins, pharmaceuticals, animal feed supplements, specialtychemicals, chemical feedstocks, plastics, solvents, fuels, or otherorganic polymers, lactic acid, and ethanol, including fuel ethanol. Inparticular the sugars may be used as feedstocks for fermentation intochemicals, plastics, such as for instance succinic acid and (bio) fuels,including ethanol, methanol, butanol synthetic liquid fuels and biogas.

For instance, in the method of the invention, an enzyme or combinationof enzymes acts on a lignocellulosic substrate or plant biomass, servingas the feedstock, so as to convert this complex substrate to simplesugars and oligosaccharides for the production of ethanol or otheruseful fermentation products.

Sugars released from biomass can be converted to useful fermentationproducts such a one of those including, but not limited to, amino acids,vitamins, pharmaceuticals, animal feed supplements, specialty chemicals,chemical feedstocks, plastics, and ethanol, including fuel ethanol.

Accordingly, the invention provides a method for the preparation of afermentation product, which method comprises:

a. degrading lignocellulose using a method as described herein; and

b. fermentation of the resulting material,

thereby to prepare a fermentation product.

The fermentation may be carried out under aerobic or anaerobicconditions. Preferably, the process is carried out undermicro-aerophilic or oxygen limited conditions.

An anaerobic fermentation process is herein defined as a fermentationprocess run in the absence of oxygen or in which substantially no oxygenis consumed, preferably about 5 or less, about 2.5 or less or about 1mmol/L/h or less, and wherein organic molecules serve as both electrondonor and electron acceptors.

An oxygen-limited fermentation process is a process in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The degree of oxygen limitation is determined by the amount andcomposition of the ingoing gas flow as well as the actual mixing/masstransfer properties of the fermentation equipment used. Preferably, in aprocess under oxygen-limited conditions, the rate of oxygen consumptionis at least about 5.5, more preferably at least about 6 and even morepreferably at least about 7 mmol/L/h.

A method for the preparation of a fermentation product may optionallycomprise recovery of the fermentation product.

SSF

Fermentation and Saccharification may also be executed in SimultaneousSaccharification and Fermentation (SSF) mode. One of the advantages ofthis mode is reduction of the sugar inhibition on enzymatic hydrolysis(Sugar inhibition on cellulases is described by Caminal B&B Vol XXVII Pp1282-1290).

Fermentation Products

Fermentation products which may be produced according to the inventioninclude amino acids, vitamins, pharmaceuticals, animal feed supplements,specialty chemicals, chemical feedstocks, plastics, solvents, fuels, orother organic polymers, lactic acid, and ethanol, including fuel ethanol(the term “ethanol” being understood to include ethyl alcohol ormixtures of ethyl alcohol and water).

Specific value-added products that may be produced by the methods of theinvention include, but not limited to, biofuels (including ethanol andbutanol and a biogas); lactic acid; a plastic; a specialty chemical; anorganic acid, including citric acid, succinic acid, fumaric acid,itaconic acid and maleic acid; 3-hydoxy-propionic acid, acrylic acid;acetic acid; 1,3-propane-diol; ethylene, glycerol; a solvent; an animalfeed supplement; a pharmaceutical, such as a β-lactam antibiotic or acephalosporin; vitamins; an amino acid, such as lysine, methionine,tryptophan, threonine, and aspartic acid; an industrial enzyme, such asa protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, alyase, an oxidoreductases, a transferase or a xylanase; and a chemicalfeedstock.

Biogas

The invention also provides use of a polypeptide or composition adescribed herein in a method for the preparation of biogas. Biogastypically refers to a gas produced by the biological breakdown oforganic matter, for example non-starch carbohydrate containing material,in the absence of oxygen. Biogas originates from biogenic material andis a type of biofuel. One type of biogas is produced by anaerobicdigestion or fermentation of biodegradable materials such as biomass,manure or sewage, municipal waste, and energy crops. This type of biogasis comprised primarily of methane and carbon dioxide. The gas methanecan be combusted or oxidized with oxygen. Air contains 21% oxygen. Thisenergy release allows biogas to be used as a fuel. Biogas can be used asa low-cost fuel in any country for any heating purpose, such as cooking.It can also be utilized in modern waste management facilities where itcan be used to run any type of heat engine, to generate eithermechanical or electrical power.

The first step in microbial biogas production consists in the enzymaticdegradation of polymers and complex substrates (for example non-starchcarbohydrate). Accordingly, the invention provides a method forpreparation of a biogas in which a substrate comprising non-starchcarbohydrate is contacted with a polypeptide or composition of theinvention, thereby to yield fermentable material which may be convertedinto a biogas by an organism such as a microorganism. In such a method,a polypeptide of the invention may be provided by way of an organism,for example a microorganism which expresses such a polypeptide.

Use of Enzymes in Food Products

The polypeptides and compositions of the invention may be used in amethod of processing plant material to degrade or modify the celluloseor hemicellulose or pectic substance constituents of the cell walls ofthe plant or fungal material. Such methods may be useful in thepreparation of food product. Accordingly, the invention provides amethod for preparing a food product which method comprises incorporatinga polypeptide or composition of the invention during preparation of thefood product.

The invention also provides a method of processing a plant materialwhich method comprises contacting the plant material with a polypeptideor composition of the invention to degrade or modify the cellulose inthe (plant) material. Preferably, the plant material is a plant pulp orplant extract, such as juices.

The present invention also provides a method for reducing the viscosity,clarity and/or filterability of a plant extract which method comprisescontacting the plant extract with a polypeptide or composition of theinvention in an amount effective in degrading cellulose or hemicelluloseor pectic substances contained in the plant extract.

Plant and cellulose/hemicellulose/pectic substance-containing materialsinclude plant pulp, parts of plants and plant extracts. In the contextof this invention an extract from a plant material is any substancewhich can be derived from plant material by extraction (mechanicaland/or chemical), processing or by other separation techniques. Theextract may be juice, nectar, base, or concentrates made thereof. Theplant material may comprise or be derived from vegetables, e.g.,carrots, celery, onions, legumes or leguminous plants (soy, soybean,peas) or fruit, e.g., pome or seed fruit (apples, pears, quince etc.),grapes, tomatoes, citrus (orange, lemon, lime, mandarin), melons,prunes, cherries, black currants, redcurrants, raspberries,strawberries, cranberries, pineapple and other tropical fruits, treesand parts thereof (e.g. pollen, from pine trees), or cereal (oats,barley, wheat, maize, rice). The material (to be hydrolysed) may also beagricultural residues, such as sugar beet pulp, corn cobs, wheat straw,(ground) nutshells, or recyclable materials, e.g. (waste) paper.

The polypeptides of the invention can thus be used to treat plantmaterial including plant pulp and plant extracts. They may also be usedto treat liquid or solid foodstuffs or edible foodstuff ingredients, orbe used in the extraction of coffee, plant oils, starch or as athickener in foods.

Typically, the polypeptides of the invention are used as acomposition/enzyme preparation as described above. The composition willgenerally be added to plant pulp obtainable by, for example mechanicalprocessing such as crushing or milling plant material. Incubation of thecomposition with the plant will typically be carried out for at time offrom 10 minutes to 5 hours, such as 30 minutes to 2 hours, preferablyfor about 1 hour. The processing temperature is preferably from about10° C. to about 55° C., e.g. from about 15° C. to about 25° C.,optimally about 20° C. and one can use from about 10 g to about 300 g,preferably from about 30 g to about 70 g, optimally about 50 g of enzymeper ton of material to be treated.

All of the enzyme(s) or their compositions used may be addedsequentially or at the same time to the plant pulp. Depending on thecomposition of the enzyme preparation the plant material may first bemacerated (e.g. to a pure) or liquefied. Using the polypeptides of theinvention processing parameters such as the yield of the extraction,viscosity of the extract and/or quality of the extract can be improved.

Alternatively, or in addition to the above, a polypeptide of theinvention may be added to the raw juice obtained from pressing orliquefying the plant pulp. Treatment of the raw juice will be carriedout in a similar manner to the plant pulp in respect of dosage,temperature and holding time. Again, other enzymes such as thosediscussed previously may be included. Typical incubation conditions areas described in the previous paragraph.

Once the raw juice has been incubated with the polypeptides of theinvention, the juice is then centrifuged or (ultra) filtered to producethe final product.

After treatment with the polypeptide of the invention the (end) productcan be heat treated, e.g. at about 100° C. for a time of from about 1minute to about 1 hour, under conditions to partially or fullyinactivate the polypeptide(s) of the invention.

A composition containing a polypeptide of the invention may also be usedduring the preparation of fruit or vegetable purees.

The polypeptide of the invention may also be used in brewing, winemaking, distilling or baking. It may therefore be used in thepreparation of alcoholic beverages such as wine and beer. For example itmay improve the filterability or clarity, for example of beers, wort(e.g. containing barley and/or sorghum malt) or wine.

Furthermore, a polypeptide or composition of the invention may be usedfor treatment of brewers spent grain, i.e. residuals from beer wortproduction containing barley or malted barley or other cereals, so as toimprove the utilization of the residuals for, e.g., animal feed.

The protein may assist in the removal of dissolved organic substancesfrom broth or culture media, for example where distillery waste fromorganic origin is bioconverted into microbial biomass. The polypeptideof the invention may improve filterability and/or reduce viscosity inglucose syrups, such as from cereals produced by liquefaction (e.g. withα-amylase).

In baking the polypeptide may improve the dough structure, modify itsstickiness or suppleness, improve the loaf volume and/or crumb structureor impart better textural characteristics such as break, shred or crumbquality.

The present invention thus relates to methods for preparing a dough or acereal-based food product comprising incorporating into the dough apolypeptide or composition of the present invention. This may improveone or more properties of the dough or the cereal-based food productobtained from the dough relative to a dough or a cereal-based foodproduct in which the polypeptide is not incorporated.

The preparation of the cereal-based food product according to theinvention further can comprise steps known in the art such as boiling,drying, frying, steaming or baking of the obtained dough.

Products that are made from a dough that is boiled are for exampleboiled noodles, dumplings, products that are made from fried dough arefor example doughnuts, beignets, fried noodles, products that are madefor steamed dough are for example steamed buns and steamed noodles,examples of products made from dried dough are pasta and dried noodlesand examples of products made from baked dough are bread, cookies andcake.

The term “improved property” is defined herein as any property of adough and/or a product obtained from the dough, particularly acereal-based food product, which is improved by the action of thepolypeptide according to the invention relative to a dough or product inwhich the polypeptide according to the invention is not incorporated.The improved property may include, but is not limited to, increasedstrength of the dough, increased elasticity of the dough, increasedstability of the dough, improved machinability of the dough, improvedproofing resistance of the dough, reduced stickiness of the dough,improved extensibility of the dough, increased volume of thecereal-based food product, reduced blistering of the cereal-based foodproduct, improved crumb structure of the baked product, improvedsoftness of the cereal-based food product, improved flavour of thecereal-based food product, improved anti-staling of the cereal-basedfood product. Improved properties related to pasta and noodle type ofcereal-based products are for example improved firmness, reducedstickiness, improved cohesiveness and reduced cooking loss.

The improved property may be determined by comparison of a dough and/ora cereal-based food product prepared with and without addition of apolypeptide of the present invention. Organoleptic qualities may beevaluated using procedures well established in the baking industry, andmay include, for example, the use of a panel of trained taste-testers.

The term “dough” is defined herein as a mixture of cereal flour andother ingredients firm enough to knead or roll. Examples of cereals arewheat, rye, corn, maize, barley, rice, groats, buckwheat and oat. Wheatis I here and hereafter intended to encompass all known species ofTriticum genus, for example aestivum, durum and/or spelt. Examples ofsuitable other ingredients are: the cellobiohydrolase according to thepresent invention, additional enzymes, chemical additives and/orprocessing aids. The dough may be fresh, frozen, pre-pared, orpre-baked. The preparation of a dough from the ingredients describedabove is well known in the art and comprises mixing of said ingredientsand processing aids and one or more moulding and optionally fermentationsteps. The preparation of frozen dough is described by Kulp and Lorenzin Frozen and Refrigerated Doughs and Batters.

The term “cereal-based food product” is defined herein as any productprepared from a dough, either of a soft or a crisp character. Examplesof cereal-based food products, whether of a white, light or dark type,which may be advantageously produced by the present invention are bread(in particular white, whole-meal or rye bread), typically in the form ofloaves or rolls, French baguette-type bread, pasta, noodles, doughnuts,bagels, cake, pita bread, tortillas, tacos, cakes, pancakes, biscuits,cookies, pie crusts, steamed bread, and crisp bread, and the like.

The term “baked product” is defined herein as any cereal-based foodproduct prepared by baking the dough.

Non-starch polysaccharides (NSP) can increase the viscosity of thedigesta which can, in turn, decrease nutrient availability and animalperformance. The use of the cellobiohydrolase of the present inventioncan improve phosphorus utilization as well as cation minerals andprotein during animal digesta.

Adding specific nutrients to feed improves animal digestion and therebyreduces feed costs. A lot of feed additives are being currently used andnew concepts are continuously developed. Use of specific enzymes likenon-starch carbohydrate degrading enzymes could breakdown the fibrereleasing energy as well as increasing the protein digestibility due tobetter accessibility of the protein when the fibre gets broken down. Inthis way the feed cost could come down as well as the protein levels inthe feed also could be reduced.

Non-starch polysaccharides (NSPs) are also present in virtually all feedingredients of plant origin. NSPs are poorly utilized and can, whensolubilized, exert adverse effects on digestion. Exogenous enzymes cancontribute to a better utilization of these NSPs and as a consequencereduce any anti-nutritional effects. Non-starch carbohydrate degradingenzymes of the present invention can be used for this purpose incereal-based diets for poultry and, to a lesser extent, for pigs andother species.

A non-starch carbohydrate degrading polypeptide/enzyme of the invention(of a composition comprising the polypeptide/enzyme of the invention)may be used in the detergent industry, for example for removal fromlaundry of carbohydrate-based stains. A detergent composition maycomprise a polypeptide/enzyme of the invention and, in addition, one ormore of a cellulase, a hemicellulase, a pectinase, a protease, a lipase,a cutinase, an amylase or a carbohydrase.

Use of Enzymes in Detergent Compositions

A detergent composition comprising an a polypeptide or composition ofthe invention may be in any convenient form, for example a paste, a gel,a powder or a liquid. A liquid detergent may be aqueous, typicallycontaining up to about 70% water and from about 0 to about 30% organicsolvent or non-aqueous material.

Such a detergent composition may, for example, be formulated as a handor machine laundry detergent composition including a laundry additivecomposition suitable for pre-treatment of stained fabrics and a rinseadded fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dish washingoperations.

In general, the properties of the enzyme should be compatible with theaselected detergent (for example, pH-optimum, compatibility with otherenzymatic and/or non-enzymatic ingredients, etc.) and the enzyme(s)should be present in an effective amount.

A detergent composition may comprise a surfactant, for example ananionic or non-ionic surfactant, a detergent builder or complexingagent, one or more polymers, a bleaching system (for example an H₂O₂source) or an enzyme stabilizer. A detergent composition may alsocomprise any other conventional detergent ingredient such as, forexample, a conditioner including a clay, a foam booster, a sudsuppressor, an anti-corrosion agent, a soil-suspending agent, an an-soilredeposition agent, a dye, a bactericide, an optical brightener, ahydrotropes, a tarnish inhibitor or a perfume.

Use of Enzymes in Paper and Pulp Processing

A polypeptide or composition of the present invention may be used in thepaper and pulp industry, inter alia in the bleaching process to enhancethe brightness of bleached pulps whereby the amount of chlorine used inthe bleaching stages may be reduced, and to increase the freeness ofpulps in the recycled paper process (Eriksson, K. E. L., Wood Scienceand Technology 24 (1990):79-101; Paice, et al., Biotechnol. and Bioeng.32 (1988):235-239 and Pommier et al., Tappi Journal (1989):187-191).Furthermore, a polypeptide or composition of the invention may be usedfor treatment of lignocellulosic pulp so as to improve the bleachabilitythereof. Thereby the amount of chlorine need to obtain a satisfactorybleaching of the pulp may be reduced.

A polypeptide or composition of the invention may be used in a method ofreducing the rate at which cellulose-containing fabrics become harsh orof reducing the harshness of cellulose-containing fabrics, the methodcomprising treating cellulose-containing fabrics with a polypeptide orcomposition as described above. The present invention further relates toa method providing colour clarification of coloured cellulose-containingfabrics, the method comprising treating coloured cellulose-containingfabrics with a polypeptide or composition as described above, and amethod of providing a localized variation in colour of colouredcellulose-containing fabrics, the method comprising treating colouredcellulose-containing fabrics with a polypeptide or composition asdescribed above. The methods of the invention may be carried out bytreating cellulose-containing fabrics during washing. However, ifdesired, treatment of the fabrics may also be carried out during soakingor rinsing or simply by adding the polypeptide or composition asdescribed above to water in which the fabrics are or will be immersed.

Other Enzyme Uses

In addition, a polypeptide or composition of the present invention canalso be used in antibacterial formulation as well as in pharmaceuticalproducts such as throat lozenges, toothpastes, and mouthwash.

The following Examples illustrate the invention:

EXPERIMENTAL INFORMATION Strains and Enzyme Compositions

Aspergillus niger strain is deposited at the CBS Institute under thedeposit number CBS 513.88.

Rasamsonia (Talaromyces) emersonii strain TEC-142 is deposited atCENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167,NL-3508 AD Utrecht, The Netherlands on 1 Jul. 2009 having the AccessionNumber CBS 124902. TEC-142S is a single isolate of TEC-142.

Rasamsonia (Talaromyces) emersonii strain was deposited at CENTRAALBUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 ADUtrecht, The Netherlands in December 1964 having the Accession NumberCBS 393.64. Other suitable strains can be equally used in the presentexamples to show the effect and advantages of the invention. For exampleTEC-101, TEC-147, TEC-192, TEC-201 or TEC-210 are suitable Rasamsoniastrains which are described in WO2011/000949.

TEC-210 cellulase-containing composition was produced according to theprocedures such as inoculation and fermentation as described inWO2011/000949.

Beta-glucosidase (BG) is produced by overexpression of EBA4 inAspergillus niger as described in WO2011/098577 followed by fermentationof the Aspergillus niger transformant. EBA4 is a Rasamsonia emersonii(Talaromyces emersonii) BG and is identified in WO2011/098577 as T.emersonii beta-glucosidase (BG) and represented by SEQ ID NO: 5 inWO2011/098577.

Celluclast (Trichoderma cellulase) was obtained from Sigma

Molecular Biology Techniques

In these strains, using molecular biology techniques known to theskilled person (see: Sambrook & Russell, Molecular Cloning: A LaboratoryManual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001), severalgenes were over expressed and others were down regulated as describedbelow. Examples of the general design of expression vectors for geneover expression and disruption vectors for down-regulation,transformation, use of markers and selective media can be found inWO199846772, WO199932617, WO2001121779, WO2005095624, WO2006040312, EP635574B, WO2005100573, WO2011009700, WO2012001169 and WO2011054899. Allgene replacement vectors comprise approximately 1-2 kb flanking regionsof the respective ORF sequences, to target for homologous recombinationat the predestined genomic loci. In addition, A. niger vectors containthe A. nidulans bi-directional amdS selection marker for transformation,in-between direct repeats. The method applied for gene deletion in allexamples herein uses linear DNA, which integrates into the genome at thehomologous locus of the flanking sequences by a double cross-over, thussubstituting the gene to be deleted by the amdS gene. Aftertransformation, the direct repeats allow for the removal of theselection marker by a (second) homologous recombination event. Theremoval of the amdS marker can be done by plating on fluoro-acetamidemedia, resulting in the selection of marker-gene-free strains. Usingthis strategy of transformation and subsequent counter-selection, whichis also described as the “MARKER-GENE FREE” approach in EP 0 635 574,the amdS marker can be used indefinitely in strain modificationprograms.

Media and Solutions:

Potato Dextrose Agar, PDA, (Fluka, Cat. No. 70139): Per Litre:

Potato extrac 4 g; Dextrose 20 g; Bacto agar 15 g; pH 5.4; Sterilize 20min at 120° C.

Rasamsonia Agar Medium: Per Litre:

Salt fraction no. 3 15 g; Cellulose 30 g; Bacto peptone 7.5 g; Grainflour 15 g; KH2PO4 5 g; CaCl2.2aq 1 g; Bacto agar 20 g; pH 6.0;Sterilize 20 min at 120° C.

Salt Fraction Composition:

The “salt fraction no. 3” was fitting the disclosure of WO98/37179,Table 1. Deviations from the composition of this table were CaCl2.2aq1.0 g/l, KCl 1.8 g/L, citric acid 1 aq 0.45 g/L (chelating agent).

Shake Flask Media for Rasamsonia

Rasamsonia Medium 1: Per Litre:

Glucose 20 g; Yeast extract (Difco) 20 g; Clerol FBA3107 (AF) 4 drops;pH 6.0; Sterilize 20 min at 120° C.

Rasamsonia Medium 2: Per Litre:

Salt fraction no. 3 15 g; Cellulose 20 g; Bacto peptone 4 g; Grain flour7.5 g; KH2PO4 10 g; CaCl2.2H20 0.5 g; Clerol FBA3107 (AF) 0.4 ml; pH 5;Sterilize 20 min at 120° C.

Rasamsonia Medium 3: Per Litre:

Salt fraction no. 3 15 g; glucose 50 g; Bacto peptone 7.5 g; KH2PO4 10g; CaCl2.2H20 0.5 g; Clerol FBA3107 (AF) 0.4 ml; pH 5; Sterilize 20 minat 120° C.

Spore Batch Preparation for Rasamsonia

Strains were grown from stocks on Rasamsonia agar medium in 10 cmdiameter Petri dishes for 5-7 days at 40° C. For MTP fermentations,strains were grown in 96-well plates containing Rasamsonia agar medium.Strain stocks were stored at −80° C. in 10% glycerol.

Chromosomal DNA Isolation

Strains were grown in YGG medium (per liter: 8 g KCl, 16 g glucose.H2O,20 ml of 10% yeast extract, 10 ml of 100× pen/strep, 6.66 g YNB+aminoacids, 1.5 g citric acid, and 6 g K2HPO4). for 16 hours at 42° C., 250rpm, and chromosomal DNA was isolated using the DNeasy plant mini kit(Qiagen, Hilden, Germany).

MTP Fermentation of Rasamsonia

96 wells microtiter plates (MTP) with sporulated R. emersonii strainswere used to harvest spores for MTP fermentations. To do this, 200 μl of10 times diluted Rasamsonia medium 1 was added to each well and afterresuspending the mixture, 100 μl of spore suspension was incubated inhumidity shakers (Infors) for 44° C. at 550 rpm, and 80% humidity for 16hours. Subsequently, 50 μl of pre-culture was used to inoculate 250 μlof Rasamsonia medium 2 in MTP plates. The 96-well plates were incubatedin humidity shakers (Infors) for 44° C. at 550 rpm, and 80% humidity for6 days. Plates were centrifuged and supernatants were harvested.

Shake Flask Growth Protocol of Rasamsonia

Spores were directly inoculated into 500 ml shake flasks containing 100ml of either Rasamsonia medium 2 or 3 and incubated at 45° C. at 250 rpmin an incubator shaker for 3-4 days. Alternatively, spores wereinoculated in 100 ml shake flasks containing Rasamsonia medium 1 andincubated at 45° C. at 250 rpm in an incubator shaker for 1 day(preculture) and, subsequently, 5 or 10 ml of biomass from thepre-culture was transferred to 500 ml shake flasks containing 100 ml ofRasamsonia medium 2 or 3 and grown under conditions as described above.

Protein Analysis

Protein samples were separated under reducing conditions on NuPAGE 4-12%Bis-Tris gel (Invitrogen, Breda, The Netherlands) and stained. Gels werestained with either InstantBlue (Expedeon, Cambridge, United Kingdom),SimplyBlue safestain (Invitrogen, Breda, The Netherlands) or Sypro Ruby(Invitrogen, Breda, The Netherlands) according to manufacturer'sinstructions.

Total Protein Content

Protein content of the recovered supernatant was determined according toBradford method. The amount of protein in the enzyme samples wasdetermined with Bradford Protein assay, using Coomassie protein reagent.25 μl of appropriately diluted enzyme sample was mixed with 1.2 mlCoomassie reagent. After 10 minutes at room temperature the absorbanceof the mixture at 595 nm was determined using a spectrophotometer(Uvikon XL). Protein content was calculated in comparison to BSAstandard.

Sugar-Release Activity Assay from Acid Pretreated Corn Stover Feedstock

For each (hemi-)cellulase assay condition, the enzyme culturesupernatant was analysed in duplicate according to the followingprocedure: 5 mg protein/g dry matter feedstock of the enzyme culturesupernatant was transferred to a suitable vial containing 800 μL 2.5%(w/w) dry matter of a mildly acid pre-treated corn stover substrate in a50 mM citrate buffer, buffered at pH 3.5 or pH 4.5 or 5.0. Additionally,as a blank sample the same amount of enzyme culture supernatant wasadded to another vial, where the 800 μL 2.5% (w/w) dry matter of amildly acid pre-treated corn stover substrate in a 50 mM citrate bufferwas replaced by 800 μL 50 mM citrate buffer, buffered at pH 4.5. Theassay samples buffered at pH 3.5 were incubated at 65° C. for 72 hours.The assay samples buffered at pH 5.0 were incubated at 50° C. for 72hours. The assay samples buffered at pH 4.5, and blank samples forcorrection of the monomeric sugar content in the enzyme supernatantswere incubated at 65° C. for 72 hours. Also, assay samples buffered atpH 4.5 were incubated at 75° C. for 72 hours.

In addition to the individual incubations as described above, the enzymeculture supernatant was also tested in combination with two differenthemicellulase mixtures; TEC-210 (Rasamsonia emersonii) to whichadditional beta-glucosidase (BG) (Aspergillus niger strain expressing aBG from Rasamsonia emersonii) was added (0.08 mg/g dry matter) andCelluclast (Trichoderma reesei) to which additional BG (Novozym-188) wasadded (0.08 mg/g dry matter). The mixtures were added to a concentrationof 1 mg protein/g dry matter of the feedstock. These incubations wereperformed at the same conditions as described above.

For each procedure, an assay was performed where the enzyme supernatantwas replaced by demineralized water, in order to correct for possiblemonomeric sugars present in the feedstock after incubation.

After incubation of the assay samples, a fixed volume of an internalstandard, maleic acid (20 g/L), EDTA (40 g/L) and DSS(2,2-Dimethyl-2-silapentane-5-sulfonate) (0.5 g/L), was added to eachvial. After centrifugation, 650 μL of the supernatant was transferred toa new vial.

The supernatant of the samples is lyophilized overnight, subsequently 50μL D2O is added to the dried residue and lyophilized once more. Thedried residue is dissolved in 600 μL of D2O. 1D 1H NMR spectra arerecorded on a Bruker Avance III HD 400 MHz, equipped with a N2 cooledcryo-probe, using a pulse program without water suppression at atemperature of 17° C. with a 90 degrees excitation pulse, acquisitiontime of 2.0 s and relaxation delay of 10 s. The analyte concentrationsare calculated based on the following signals (δ relative to DSS(4,4-dimethyl-4-silapentane-1-sulfonic acid)): ½ of β-glucose peak at4.63 ppm (d, 0.31H, J=8 Hz), ½ of β-xylose peak at 4.56 ppm (d, 0.315H,J=8 Hz), Xylo-oligo peak at 4.45 ppm (d, 1H, J=8 Hz), ½ of β anomer ofthe reducing end of cellobiose peak at 4.66 ppm (d, 0.31H, J=8 Hz). Thesignal user for the standard: Maleic acid peak at 6.26 ppm (s, 2H)

The (hemi)-cellulase enzyme solution may contain residual sugars.Therefore, the results of the assay are corrected for the sugar contentmeasured after incubation of the enzyme solution.

β-xylosidase Activity Measurement

This assay measures the release of p-nitrophenol by the action ofβ-xylosidase on p-nitrophenyl-β-D-xylopyranoside (PNPX). Oneβ-xylosidase unit of activity is the amount of enzyme that liberates 1micromole of p-nitrophenol in one minute at 60° C. and pH 4.5. Acetatebuffer (0.1 M, pH 4.5) is prepared as follows: 8.2 g of anhydrous sodiumacetate is dissolved in distilled water so that the final volume of thesolution is 1000 ml (Solution A). In a separate flask, 6.0 g (5.72 ml)of glacial acetic acid is mixed with distilled water to make the totalvolume of 1000 ml (Solution B). The final 0.1 M acetate buffer, pH 4.5is prepared by mixing Solution A with Solution B until the pH of theresulting solution is equal to 4.5. A drop (˜25 μL) Triton X-100 isadded/L buffer solution. PNPX (Sigma) is used as the assay substrate.

100 mg of PNPX is dissolved in 84 mL of 0.1 M acetate buffer to obtain a4.4 mM stock solution. The stop reagent (1 M sodium carbonate solution)is prepared as follows: 10.6 g of anhydrous sodium carbonate isdissolved in 50 ml of distilled water, and the solution volume isadjusted to 100 ml. This reagent is used to terminate the enzymaticreaction.

For the incubation with enzyme, 0.1 mL of 4.4 mM PNPX stock solution ismixed with 0.1 mL of the appropriate diluted enzyme sample and incubatedat 60° C. for 60 minutes. After 60 minutes of incubation, 0.1 mL of thereaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solutionand the absorbance is measured at 405 nm in microtiter plates as A_(S)

For the substrate blank, 0.1 mL of 4.4 mM PNPX stock solution is mixedwith 0.1 mL of 0.1 M acetate buffer, pH 4.5 and treated the same as thesamples: incubated at 60° C. for 60 minutes after which 0.1 mL of thereaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solutionand the absorbance at 405 nm is measured in microtiter plates as A_(SB).

Enzyme blanks (without addition of substrate) are measured to correctfor background color originating from the enzymes. 0.1 mL of theappropriate diluted enzyme sample is mixed with 0.1 mL 0.1 M acetatebuffer, pH 4.5 and incubated at 60° C. for 60 minutes. After 60 minutesof incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1M sodium carbonate solution and the absorbance is measured at 405 nm inmicrotiter plates as A_(EB).

A calibration curve of p-nitrophenol (appropriate diluted in 0.1 Macetate buffer, pH 4.5) mixed in a ratio of 1:1 with 1 M sodiumcarbonate solution is used to quantify its release from PNPX by theaction of the enzyme.

After the incubation of enzyme with substrate the corrected absorbance(=A_(S)-A_(EB)-A_(SB)), is used to calculate the amount of p-nitrophenolreleased by the enzyme.

The activity is expressed as the amount of enzyme required to release 1μM p-nitrophenol/min under the assay conditions.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH3, GH30, GH39, GH43, GH52, and GH54 enzymes.

β-xylosidase activity Assay 2

This assay measures the release of xylose by the action of β-xylosidaseon xylobiose.

Sodium acetate buffer (0.05 M, pH 4.5) was prepared as follows. 4.1 g ofanhydrous sodium acetate or 6.8 g of sodium acetate*3H₂O was dissolvedin distilled water to a final volume of 1000 mL (Solution A). In aseparate flask, 3.0 g (2.86 mL) of glacial acetic acid was mixed withdistilled water to make the total volume of 1000 mL (Solution B). Thefinal 0.05 M sodium acetate buffer, pH 4.5, was prepared by mixingSolution A with Solution B until the pH of the resulting solution wasequal to 4.5. Xylobiose was purchased from Sigma and dissolved in sodiumacetate buffer pH 4.5 to a concentration of 100 ug/mL

The assay was performed as detailed below.

The enzyme culture supernatant was added to the substrate in a dosage of1 and 5 mg protein/g substrate which was then incubated at 62° C. for 24hours. The reaction was stopped by heating the samples for 10 minutes at100° C. The release of xylose was analyzed by High Performance AnionExchange Chromatography.

Substrate Blank

Instead of enzyme culture supernatant the same amount of buffer wasadded to the substrate solution which was then incubated at 62° C. for24 hours. The reaction was stopped by heating the samples for 10 minutesat 100° C. The sample was analyzed by High Performance Anion ExchangeChromatography. The analysis was performed using a Dionex HPLC systemequipped with a Dionex CarboPac PA-1 (2 mm ID×250 mm) column incombination with a CarboPac PA guard column (2 mm ID×50 mm) and a DionexPAD-detector (Dionex Co. Sunnyvale). A flow rate of 0.3 mL/min was usedwith the following gradient of sodium acetate in 0.1 M NaOH: 0-20 min,0-180 mM. Each elution was followed by a washing step of 5 min 1000 mMsodium acetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 MNaOH.

In case interfering compounds were present that complicate xyloseidentification the analysis was performed by running isocratic on H₂Ofor 30 min a gradient (0.5M NaOH was added post-column at 0.1 mL/min fordetection) followed by a washing step of 5 min 1000 mM sodium acetate in0.1 M NaOH and an equilibration step of 15 min H₂O.

Standards of xylose and xylobiose (Sigma) were used for identificationof the substrate and product formed by the enzyme.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH3, GH30, GH39, GH43, GH52, and GH54 enzymes.

β-xylosidase Activity Assay 3

The same assay as described above was performed with xylan substrateslike Oat arabinoxylan, Beech wood xylan and Birch wood xylan (Sigma)instead of xylobiose to measure xylosidase activity on polymericsubstrates.

Assay conditions were the same with the exception that all substrateswere solved to a concentration of 2 mg/mL. The incubation was performedat 60° C. for 24 h at a dosage of 10 mg/g. Next to xylose and xylobiosealso xylotriose and xylotetraose were quantified.

α-galactosidase Activity Measurement

This assay measures the release of p-nitrophenol by the action ofα-galactosidase on p-nitrophenyl-α-D-galactopyranoside (PNPG). Oneα-galactosidase unit of activity is the amount of enzyme that liberates1 micromole of p-nitrophenol in one minute at 60° C. and pH 4.5. Acetatebuffer (0.1 M, pH 4.5) is prepared as follows: 8.2 g of anhydrous sodiumacetate is dissolved in distilled water so that the final volume of thesolution is 1000 ml (Solution A). In a separate flask, 6.0 g (5.72 ml)of glacial acetic acid is mixed with distilled water to make the totalvolume of 1000 ml (Solution B). The final 0.1 M acetate buffer, pH 4.5is prepared by mixing Solution A with Solution B until the pH of theresulting solution is equal to 4.5. A drop (˜25 μL) Triton X-100 isadded/L buffer solution. PNPG (Sigma) is used as the assay substrate.

A stock solution of 4.4 mM PNPG is made in 0.1 M acetate buffer. Thestop reagent (1 M sodium carbonate solution) is prepared as follows:10.6 g of anhydrous sodium carbonate is dissolved in 50 ml of distilledwater, and the solution volume is adjusted to 100 ml. This reagent isused to terminate the enzymatic reaction.

For the incubation with enzyme, 0.1 mL of 4.4 mM PNPG stock solution ismixed with 0.1 mL of the appropriate diluted enzyme sample and incubatedat 60° C. for 60 minutes. After 60 minutes of incubation, 0.1 mL of thereaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solutionand the absorbance is measured at 405 nm in microtiter plates as A_(S)

For the substrate blank, 0.1 mL of 4.4 mM PNPG stock solution is mixedwith 0.1 mL of 0.1 M acetate buffer, pH 4.5 and treated the same as thesamples: incubated at 60° C. for 60 minutes after which 0.1 mL of thereaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solutionand the absorbance at 405 nm is measured in microtiter plates as A_(SB).

Enzyme blanks (without addition of substrate) are measured to correctfor background color originating from the enzymes. 0.1 mL of theappropriate diluted enzyme sample is mixed with 0.1 mL 0.1 M acetatebuffer, pH 4.5 and incubated at 60° C. for 60 minutes. After 60 minutesof incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1M sodium carbonate solution and the absorbance is measured at 405 nm inmicrotiter plates as A_(EB).

A calibration curve of p-nitrophenol (appropriate diluted in 0.1 Macetate buffer, pH 4.5) mixed in a ratio of 1:1 with 1 M sodiumcarbonate solution is used to quantify its release from PNPG by theaction of the enzyme.

After the incubation of enzyme with substrate the corrected absorbance(=A_(S)-A_(EB)-A_(SB)), is used to calculate the amount of p-nitrophenolreleased by the enzyme.

The activity is expressed as the amount of enzyme required to release 1μM p-nitrophenol/min under the assay conditions.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH4, GH27 and GH36 enzymes.

Xyloglucanase Activity Assay 1

Sodium acetate buffer (0.05 M, pH 4.5) was prepared as follows. 4.1 g ofanhydrous sodium acetate was dissolved in distilled water to a finalvolume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) ofglacial acetic acid was mixed with distilled water to make the totalvolume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer,pH 4.5, was prepared by mixing Solution A with Solution B until the pHof the resulting solution is 4.5.

Tamarind xyloglucan was solved in sodium acetate buffer to obtain 2.0mg/mL. The enzyme culture supernatant was added to the substrate in adosage of 10 mg protein/g substrate which was then incubated at 60° C.for 24 hours. The reaction was stopped by heating the samples for 10minutes at 100° C. The release of oligosaccharides was analyzed by HighPerformance Anion Exchange Chromatography

As a blank sample the substrate was treated and incubated in the sameway but then without the addition of enzyme.As a reference the substrate was also incubated under the sameconditions with a commercial cellulase preparation from TrichodermaReesei (Celluclast; Sigma) which was diluted 50 times after which 20 μLwas added to the incubation.

The analysis was performed using a Dionex HPLC system equipped with aDionex CarboPac PA-1 (2 mm ID×250 mm) column in combination with aCarboPac PA guard column (2 mm ID×50 mm) and a Dionex PAD-detector(Dionex Co. Sunnyvale). A flow rate of 0.3 mL/min was used with thefollowing gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-150 mM.Each elution was followed by a washing step of 5 min 1000 mM sodiumacetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH5, GH12, GH16, GH44, and GH74 enzymes.

Xyloglucanase Activity Assay 2

The following example illustrates the assay to measure xyloglucanaseactivity. Such activity was demonstrated by using xyloglucan assubstrate and a reducing sugars assay (PAHBAH) as detection method. Thevalues were compared to a standard, which was prepared using acommercial cellulase preparation from Trichoderma Reesei (Celluclast;Sigma).

Reagent A: 5 g of p-Hydroxybenzoic acid hydrazide (PAHBAH) was suspendedin 60 mL water, 4.1 mL of concentrated hydrochloric acid was added andthe volume was adjusted to 100 ml. Reagent B: 24.9 g of trisodiumcitrate was dissolved in 500 ml of water. To this solution 2.2 g ofcalcium chloride and 40 g sodium hydroxide was added. The volume wasadjusted to 2 L with water. Both reagents were stored at roomtemperature. Working Reagent: 10 ml of Reagent A was added to 40 ml ofReagent B. This solution was prepared freshly every day, and was storedon ice between uses. Using the above reagents, the assay was performedas detailed below

Next to xyloglucan also carboxymethylcellulose was used as a substrateto determine the specificity of the enzyme.

After incubation 10 μl of each well was mixed with 200 μl workingreagent. These solutions were heated at 70° C. for 30. After coolingdown, the samples were analyzed by measuring the absorbance at 405 nm.Glucose was used as a standard to quantify reducing ends formed asglucose equivalents.

As controls the substrates were also incubated without addition ofenzyme culture supernatant and the enzyme culture supernatants wereincubated without substrate.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH5, GH12, GH16, GH44, and GH74 enzymes.

Xyloglucanase Activity Assay 3

Sodium acetate buffer (0.05 M, pH 4.5) is prepared as follows. 4.1 g ofanhydrous sodium acetate is dissolved in distilled water to a finalvolume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) ofglacial acetic acid is mixed with distilled water to make the totalvolume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer,pH 4.5, is prepared by mixing Solution A with Solution B until the pH ofthe resulting solution is 4.5.

Tamarind xyloglucanan is solved in sodium acetate buffer to obtain 2.0mg/mL. The enzyme is added to the substrate in a dosage of 10 mgprotein/g substrate which is then incubated at 60° C. for 24 hours. Thereaction is stopped by heating the samples for 10 minutes at 100° C. Theformation of lower molecular weight oligosaccharides is analyzed by HighPerformance size-exclusion Chromatography

As a blank sample the substrate is treated and incubated in the same waybut then without the addition of enzyme.

As a reference the substrate is also incubated under the same conditionswith a commercial cellulase preparation from e.g. Aspergillus niger orTrichoderma Reesei (the cellulase standard at its own optimaltemperature in case of inactivity at 60° C.).

The analysis is performed using High-performance size-exclusionchromatography (HPSEC) performed on three TSK-gel columns (6.0 mm×15.0cm per column) in series SuperAW4000, SuperAW3000, SuperAW2500; TosohBioscience), in combination with a PWXguard column (Tosoh Bioscience).Elution is performed at 55 C with 0.2 M sodium nitrate at 0.6 mL/min.The eluate was monitored using a Shodex RI-101 (Kawasaki) refractiveindex (RI) detector. Calibration was performed by using pullulans(Associated Polymer Labs Inc., New York, USA) with a molecular weight inthe range of 0.18-788 kDa.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH5, GH12, GH16, GH44, and GH74 enzymes.

α-arabinofuranosidase Activity Assay

The following example illustrates an assay to measure the ability ofα-arabinofuranosidases to remove the α-L-arabinofuranosyl residues fromsubstituted xylose residues.

For the complete degradation of arabinoxylans to arabinose and xylose,several enzyme activities are needed, including endo-xylanases andarabinofuranosidases. The arabinoxylan molecule from wheat is highlysubstituted with arabinosyl residues. These can be substituted either tothe C2 or the C3 position of the xylosyl residue (single substitution),or both to the C2 and C3 position of the xylose (double substitution).

Single and double substituted oligosaccharides were prepared byincubating wheat arabinoxylan (WAX; 10 mg/mL; Megazyme, Bray, Ireland)in 50 mM acetate buffer pH 4.5 with an appropriate amount ofendo-xylanase (from Aspergillus awamori, Kormelink F. et al; Journal ofBiotechnology (1993) 27: 249-265) 48 hours at 40° C. to produce ansufficient amount of arabinoxylo-oligosaccharides. The reaction wasstopped by heating the samples at 100° C. for 10 minutes. The sampleswere centrifuged for 5 minutes at 10.000×g. The supernatant was used forfurther experiments. Degradation of the arabinoxylan was followed byanalysis of the formed reducing sugars and High Performance AnionExchange Chromatography (HPAEC).

The enzyme culture supernatant was added to the single and doublesubstituted arabinoxylo-oligosaccharides (endo-xylanase treated WAX; 2mg/mL) in a dosage of 10 mg protein/g substrate in 50 mM sodium acetatebuffer which was then incubated at 65° C. for 24 hours. The reaction wasstopped by heating the samples at 100° C. for 10 minutes. The sampleswere centrifuged for 5 minutes at 10.000×g. The release of arabinose wasfollowed by HPAEC analysis.

The analysis was performed using a Dionex HPLC system equipped with aDionex CarboPac PA-1 (2 mm ID×250 mm) column in combination with aCarboPac PA guard column (2 mm ID×50 mm) and a Dionex PAD-detector(Dionex Co. Sunnyvale). A flow rate of 0.3 mL/min was used with thefollowing gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM.Each elution was followed by a washing step of 5 min 1000 mM sodiumacetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.Arabinose release was identified and quantified by a standard (Sigma).

This assay can be used to test the activity of enzymes such as, but notlimited to, GH3, GH43, GH51, GH54, and GH62 enzymes.

Endo-xylanase Activity Assay

Endo-xylanases are enzyme able to hydrolyze β-1,4 bond in the xylanbackbone, producing short xylooligosaccharides. This assay measures therelease of xylose and xylo-oligosaccharides by the action of xylanaseson wheat arabinoxylan (WAX) (Megazyme, Medium viscosity 29 cSt), Oatarabinoxylan, Beech wood xylan and Birch wood xylan (Sigma).

Sodium acetate buffer (0.05 M, pH 4.5) was prepared as follows; 4.1 g ofanhydrous sodium acetate was dissolved in distilled water to a finalvolume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) ofglacial acetic acid was mixed with distilled water to make the totalvolume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer,pH 4.5, was prepared by mixing Solution A with Solution B until the pHof the resulting solution was 4.5. Each substrate was solved in sodiumacetate buffer to obtain 2.0 mg/mL. The enzyme culture supernatant wasadded to the substrate in a dosage of 10 mg protein/g substrate whichwas then incubated at 60° C. for 20 hours. The reaction was stopped byheating the samples for 10 minutes at 100° C. The release of xylose andxylooligosaccharides was analyzed by High Performance Anion ExchangeChromatography.

As a blank sample the substrate was treated and incubated in the sameway but then without the addition of enzyme.

The analysis was performed using a Dionex HPLC system equipped with aDionex CarboPac PA-1 (2 mm ID×250 mm) column in combination with aCarboPac PA guard column (2 mm ID×50 mm) and a Dionex PAD-detector(Dionex Co. Sunnyvale). A flow rate of 0.3 mL/min was used with thefollowing gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM.Each elution was followed by a washing step of 5 min 1000 mM sodiumacetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.Standards of xylose, xylobiose and xylotriose (Sigma) were used toidentify these oligomers released by the action of the enzyme.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH5, GH8, GH10, and GH11.

α/β-xylosidase Activity Measurement

This assay measures the release of p-nitrophenol by the action ofα/β-xylosidase on p-nitrophenyl-α/β-D-xylopyranoside (PNPX). Oneβ-xylosidase unit of activity is the amount of enzyme that liberates 1micromole of p-nitrophenol in one minute at 60° C. and pH 4.5. Acetatebuffer (0.1 M, pH 4.5) is prepared as follows: 8.2 g of anhydrous sodiumacetate is dissolved in distilled water so that the final volume of thesolution is 1000 ml (Solution A). In a separate flask, 6.0 g (5.72 ml)of glacial acetic acid is mixed with distilled water to make the totalvolume of 1000 ml (Solution B). The final 0.1 M acetate buffer, pH 4.5is prepared by mixing Solution A with Solution B until the pH of theresulting solution is equal to 4.5. A drop (˜25 μL) Triton X-100 isadded/L buffer solution. PNPX (Sigma) is used as the assay substrate.

100 mg of PNPX is dissolved in 84 mL of 0.1 M acetate buffer to obtain a4.4 mM stock solution. The stop reagent (1 M sodium carbonate solution)is prepared as follows: 10.6 g of anhydrous sodium carbonate isdissolved in 50 ml of distilled water, and the solution volume isadjusted to 100 ml. This reagent is used to terminate the enzymaticreaction.

For the incubation with enzyme, 0.1 mL of 4.4 mM PNPX stock solution ismixed with 0.1 mL of the appropriate diluted enzyme sample and incubatedat 60° C. for 60 minutes. After 60 minutes of incubation, 0.1 mL of thereaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solutionand the absorbance is measured at 405 nm in microtiter plates as A_(S)

For the substrate blank, 0.1 mL of 4.4 mM PNPX stock solution is mixedwith 0.1 mL of 0.1 M acetate buffer, pH 4.5 and treated the same as thesamples: incubated at 60° C. for 60 minutes after which 0.1 mL of thereaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solutionand the absorbance at 405 nm is measured in microtiter plates as A_(SB).

Enzyme blanks (without addition of substrate) are measured to correctfor background color originating from the enzymes. 0.1 mL of theappropriate diluted enzyme sample is mixed with 0.1 mL 0.1 M acetatebuffer, pH 4.5 and incubated at 60° C. for 60 minutes. After 60 minutesof incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1M sodium carbonate solution and the absorbance is measured at 405 nm inmicrotiter plates as A_(EB).

A calibration curve of p-nitrophenol (appropriate diluted in 0.1 Macetate buffer, pH 4.5) mixed in a ratio of 1:1 with 1 M sodiumcarbonate solution is used to quantify its release from PNPX by theaction of the enzyme.

After the incubation of enzyme with substrate the corrected absorbance(=A_(S)-A_(EB)-A_(SB)), is used to calculate the amount of p-nitrophenolreleased by the enzyme.

The activity is expressed as the amount of enzyme required to release 1μM p-nitrophenol/min under the assay conditions.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH3, GH30, GH31, GH39, GH43, GH52, and GH54 enzymes.

α/β-mannosidase Activity Measurement

This assay measures the release of p-nitrophenol by the action ofα/β-mannosidase on p-nitrophenyl-α/β-D-mannopyranoside (PNPM). Oneα/β-mannosidase unit of activity is the amount of enzyme that liberates1 micromole of p-nitrophenol in one minute at 60° C. and pH 4.5. Acetatebuffer (0.1 M, pH 4.5) is prepared as follows: 8.2 g of anhydrous sodiumacetate is dissolved in distilled water so that the final volume of thesolution is 1000 ml (Solution A). In a separate flask, 6.0 g (5.72 ml)of glacial acetic acid is mixed with distilled water to make the totalvolume of 1000 ml (Solution B). The final 0.1 M acetate buffer, pH 4.5is prepared by mixing Solution A with Solution B until the pH of theresulting solution is equal to 4.5. A drop (˜25 μL) Triton X-100 isadded/L buffer solution. PNPM (Sigma) is used as the assay substrate.

A stock solution of 4.4 mM PNPM is made in 0.1 M acetate buffer. Thestop reagent (1 M sodium carbonate solution) is prepared as follows:10.6 g of anhydrous sodium carbonate is dissolved in 50 ml of distilledwater, and the solution volume is adjusted to 100 ml. This reagent isused to terminate the enzymatic reaction.

For the incubation with enzyme, 0.1 mL of 4.4 mM PNPM stock solution ismixed with 0.1 mL of the appropriate diluted enzyme sample and incubatedat 60° C. for 60 minutes. After 60 minutes of incubation, 0.1 mL of thereaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solutionand the absorbance is measured at 405 nm in microtiter plates as A_(S)

For the substrate blank, 0.1 mL of 4.4 mM PNPM stock solution is mixedwith 0.1 mL of 0.1 M acetate buffer, pH 4.5 and treated the same as thesamples: incubated at 60° C. for 60 minutes after which 0.1 mL of thereaction mixture is mixed with 0.1 mL of 1 M sodium carbonate solutionand the absorbance at 405 nm is measured in microtiter plates as A_(SB).

Enzyme blanks (without addition of substrate) are measured to correctfor background color originating from the enzymes. 0.1 mL of theappropriate diluted enzyme sample is mixed with 0.1 mL 0.1 M acetatebuffer, pH 4.5 and incubated at 60° C. for 60 minutes. After 60 minutesof incubation, 0.1 mL of the reaction mixture is mixed with 0.1 mL of 1M sodium carbonate solution and the absorbance is measured at 405 nm inmicrotiter plates as A_(EB).

A calibration curve of p-nitrophenol (appropriate diluted in 0.1 Macetate buffer, pH 4.5) mixed in a ratio of 1:1 with 1 M sodiumcarbonate solution is used to quantify its release from PNPM by theaction of the enzyme.

After the incubation of enzyme with substrate the corrected absorbance(=A_(S)-A_(EB)-A_(SB)), is used to calculate the amount of p-nitrophenolreleased by the enzyme.

The activity is expressed as the amount of enzyme required to release 1μM p-nitrophenol/min under the assay conditions.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH1, GH2, GH5, GH38, GH47, GH92, and GH125 enzymes.

Ferulovi Esterase Activity Measurement

Synthetic Substrates:

Methyl caffeate, methyl coumarate, methyl sinapinate and methyl ferulateare obtained from Apin Chemicals. Activity towards these syntheticsubstrates is determined by incubating the enzyme with the substrate ata dosage of about 5 mg/g DM at a pH of 5.0 (50 mM sodium acetatebuffer). The reaction will be done at 60° C. for up to 24 h.

At the end of the incubation the samples are boiled for 5 minutes toinactivate the enzymes and centrifuged at room temperature (10 min,10,000×g). Hydroxycinnamic acid release from the substrate is measuredby RP-UHPLC-MS analysis in negative ion mode as described earlier(Appeldoorn et al., 2010) on an Accela UHPLC system (Thermo Scientific)equipped with a Hypersyl GOLD column (2.1 mm×150 mm, 1.9 μm particlesize; Thermo Scientific). The mobile phase is composed of (A) H₂O+1%(v/v) acetonitrile+0.2% (v/v) acetic acid and (B) acetonitrile+0.2%(v/v) acetic acid. The flow rate is 0.4 mL/min, and the columntemperature is 30° C. The elution profile is as follows: first 5 min,isocratic 0% B; 5-23 min, linear from 0 to 50% B; 23-24 min, linear from50 to 100% B; 24-27 min, isocratic at 100% B; 27-28 min, linear from 100to 0% B, followed by reconditioning of the column for 7 min. Spectraldata are collected from 200 to 600 nm, and quantification is performedat 320 nm. Ferulic, caffeic, sinapic and coumaric acid contents areidentified and quantified on the basis of standards.

MS data are collected in the negative mode with an ion spray voltage of3.5 kV, a capillary voltage of −20 V, and a capillary temperature of 350C. Full MS scans are made within the range m/z 150-1500, and MS2 data ofthe most intense ions is obtained.

This assay can be used to test the activity of enzymes such as, but notlimited to, CE1 enzymes.

Feruloyl Esterase Activity Measurement

Natural Occurring Substrate:

Arabinoxylan oligomers purified from pretreated corn fibre (CF) (1 mg/mleach) (Appeldoorn et al 2010) are incubated with ferulic acid esterasesat a dosage of about 5 mg/g DM at a pH of 5.0 (50 mM sodium acetatebuffer). The reaction will be done at 60° C. for up to 24 h.

At the end of the incubation the samples are boiled for 5 minutes toinactivate the enzymes and centrifuged at room temperature (10 min,10,000×g). Hydroxycinnamic acid release from the substrate is measuredby RP-UHPLC-MS analysis in negative ion mode as described earlier(Appeldoorn et al., 2010) on an Accela UHPLC system (Thermo Scientific)equipped with a Hypersyl GOLD column (2.1 mm×150 mm, 1.9 μm particlesize; Thermo Scientific). The mobile phase is composed of (A) H₂O+1%(v/v) acetonitrile+0.2% (v/v) acetic acid and (B) acetonitrile+0.2%(v/v) acetic acid. The flow rate is 0.4 mL/min, and the columntemperature is 30° C. The elution profile is as follows: first 5 min,isocratic 0% B; 5-23 min, linear from 0 to 50% B; 23-24 min, linear from50 to 100% B; 24-27 min, isocratic at 100% B; 27-28 min, linear from 100to 0% B, followed by reconditioning of the column for 7 min. Spectraldata are collected from 200 to 600 nm, and quantification is performedat 320 nm. Ferulic and coumaric acid contents are identified andquantified on the basis of standards.

MS data are collected in the negative mode with an ion spray voltage of3.5 kV, a capillary voltage of −20 V, and a capillary temperature of 350C. Full MS scans are made within the range m/z 150-1500, and MS2 data ofthe most intense ions is obtained.

The total amount of ester-linked ferulic acid in corn oligomers wasdetermined after alkaline hydrolysis and ethylether extraction using theUHPLC method described above.

Reference:

MAAIKE M. APPELDOORN et al, J. Agric. Food Chem. 2010, 58, 11294-11301

This assay can be used to test the activity of enzymes such as, but notlimited to, CE1 enzymes.

α-glucuronidase Activity Assay

The following example illustrates the assay to measure theα-glucuronidase activity towards aldouronic acids (megazyme). This assaymeasures the release of xylose and xylooligomers by the action of theα-glucuronidase on the glucuronoxylan oligosaccharides.

Sodium acetate buffer (0.05 M, pH 4.5) was prepared as follows. 4.1 g ofanhydrous sodium acetate was dissolved in distilled water to a finalvolume of 1000 mL (Solution A). In a separate flask, 3.0 g (2.86 mL) ofglacial acetic acid was mixed with distilled water to make the totalvolume of 1000 mL (Solution B). The final 0.05 M sodium acetate buffer,pH 4.5, was prepared by mixing Solution A with Solution B until the pHof the resulting solution was 4.5.

To determine the activity on small oligomers the aldouronic acids aresolved in sodium acetate buffer to obtain 1.0 mg/mL. The enzyme culturesupernatant was added to the substrate in a dosage of 1 and 10 mgprotein/g substrate which was then incubated at 60° C. for 24 hours. Thereaction was stopped by heating the samples for 10 minutes at 100° C.The release of xylooligomers as a result of the removal of 4-O-methylglucuronic acid were analyzed by High Performance Anion ExchangeChromatography

As a blank sample the substrate was treated and incubated in the sameway but then without the addition of enzyme.

The analysis was performed using a Dionex HPLC system equipped with aDionex CarboPac PA-1 (2 mm ID×250 mm) column in combination with aCarboPac PA guard column (2 mm ID×50 mm) and a Dionex PAD-detector(Dionex Co. Sunnyvale). A flow rate of 0.3 mL/min was used with thefollowing gradient of sodium acetate in 0.1 M NaOH: 0-40 min, 0-400 mM.Each elution was followed by a washing step of 5 min 1000 mM sodiumacetate in 0.1 M NaOH and an equilibration step of 15 min 0.1 M NaOH.

Standards of xylose, xylobiose and xylotriose (Sigma) were used toidentify the xylooligomers released by the action of the enzyme thatremoves 4-O-methyl-Glucuronic acid from these oligomers.

This assay can be used to test the activity of enzymes such as, but notlimited to, GH67 and GH115 enzymes.

Example 1: Construction of A. niger Expression Vectors

This Example describes the construction of an expression construct foroverexpression of Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 in A.niger. Genomic DNA of Rasamsonia emersonii strain CBS393.64 wassequenced and analysed. The gene with translated protein annotated asactivity according in Table 1 was identified. Sequences of the R.emersonii Temer00088, Temer09484, Temer08028, Temer02362, Temer08862,Temer04790, Temer05249, Temer06848, Temer02056, Temer03124, Temer09491,Temer06400, Temer08570, Temer08163 and Temer07305 gene, comprising thecodon-pair optimised ORF sequence, protein sequence, signal sequence,genomic sequence and wild-type cDNA sequence are shown in sequencelistings SEQ ID NO: 1 to 75.

Construction of Expression Plasmids

The sequence having SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51,56, 61, 66 or 71 is cloned into the pGBTOP vector (FIG. 1) using EcoRIand PacI sites, comprising the glucoamylase promoter and terminatorsequence. The E. coli part was removed by NotI digestion prior totransformation of A. niger CBS 513.88.

Transformation of A. niger and Shake Flask Fermentations

A. niger strain CBS513.88 is co-transformed with the expressionconstructs and an appropriate selection marker (amdS or phleomycin)containing plasmid according to method described in the experimentalinformation section. Of recombinant and control A. niger strains a largebatch of spores is generated by plating spores or mycelia onto PDAplates (Potato Dextrose Agar, Oxoid), prepared according tomanufacturer's instructions. After growth for 3-7 days at 30 degreesCelsius, spores are collected after adding 0.01% Triton X-100 to theplates. After washing with sterile water about 10⁷ spores of selectedtransformants and control strains are inoculated into 100 ml shakeflasks with baffles containing 20 ml of liquid pre-culture mediumconsisting of per liter: 30 g maltose.H₂O; 5 g yeast extract; 10 ghydrolyzed casein; 1 g KH2PO4; 0.5 g MgSO₄.7H₂O; 0.03 g ZnCl₂; 0.02 gCaCl₂; 0.01 g MnSO₄.4H₂O; 0.3 g FeSO₄.7H₂O; 3 g Tween 80; 10 mlpenicillin (5000 IU/ml)/Streptomycin (5000 UG/ml); pH5.5. These culturesare grown at 34 degrees Celsius for 16-24 hours. 10 ml of this culturewas inoculated into 500 ml shake flasks with baffles containing 100 mlfermentation medium consisting of per liter: 70 g glucose.H₂O; 25 ghydrolyzed casein; 12.5 g yeast extract; 1 g KH₂PO₄; 2 g K₂SO₄; 0.5 gMgSO₄.7H₂O; 0.03 g ZnCl₂; 0.02 g CaCl₂; 0.01 g MnSO₄.4H₂O; 0.3 gFeSO₄.7H₂O; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml);adjusted to pH5.6. These cultures are grown at 34 degrees Celsius untilall glucose was depleted (usually after 4-7 days). Samples taken fromthe fermentation broth are centrifuged (10 min at 5000×g) in a swingingbucket centrifuge and supernatants collected and filtered over a 0.2 μmfilter (Nalgene)

Supernatants are analysed for expression of Temer00088, Temer09484,Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848,Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163and Temer07305 by SDS-PAGE and total protein measurements.

Example 2: Construction of a R. emersonii Expression Vectors

This Example describes the construction of an expression construct foroverexpression Temer00088, Temer09484, Temer08028, Temer02362,Temer08862, Temer04790, Temer05249, Temer06848, Temer02056, Temer03124,Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305 in R.emersonii. The expression cassette was targeted integrated into theRePepA locus.

In order to target the promoter-reporter constructs into the pepA locus,expression vectors were cloned for targeting. The gene with translatedprotein annotated as protease pepA was identified in the genome.Sequences of Rasamsonia emersonii pepA (RePepA), comprising the genomicsequence of the ORF and approximately 3000 bp of the 5′ region and 2500bp of the 3′ flanking regions, cDNA and protein sequence, are shown insequence listings 76, 77 and 78, respectively.

Two vectors were constructed according to routine cloning procedures fortargeting into the RePepA locus. The insert fragments of both vectorstogether can be applied in the so-called “bipartite gene-targeting”method (Nielsen et al., 2006, 43: 54-64). This method is using twonon-functional DNA fragments of a selection marker which are overlapping(see also WO2008113847 for further details of the bipartite method)together with gene-targeting sequences. Upon correct homologousrecombination the selection marker becomes functional by integration ata homologous target locus. As also detailed in WO 2008113847, twodifferent deletion vectors, Te pep.bbn and pEBA1006, were designed andconstructed to be able to provide the two overlapping DNA molecules forbipartite gene-targeting. The first vector Te pep.bbn (General layout asin FIG. 2) comprises a 1500 bp 5′ flanking region approximately 1.5 kbupstream of the RePepA ORF for targeting in the RePepA locus (ORF andapproximately 1500 bp of the RePepA promoter), a lox66 site, and thenon-functional 5′ part of the ble coding region driven by the A.nidulans gpdA promoter (PgpdA-ble sequence missing the last 104 bases ofthe coding sequence at the 3′ end of ble, SEQ ID NO: 79). To allowefficient cloning of promoter-reporter cassettes in E. coli, a ccdB genewas inserted in between the 5′ RePepA flanking region and the lox66site. The second pEBA1006 vector (General layout as in FIG. 3) comprisesthe non-functional 3′ part of the ble coding region and the A. nidulanstrpC terminator (ble-TtrpC sequence missing the first 12 bases of thecoding sequence at the 5′ end of ble, SEQ ID NO: 80), a lox71 site, anda 2500 bp 3′ flanking region of the RePepA ORF for targeting in theRePepA locus. Upon homologous recombination, the first and secondnon-functional fragments become functional producing a functional blecassette. Both RePepA upstream and downstream gene flanking regionstarget for homologous recombination of the bipartite fragments at thepredestined RePepA genomic locus.

The ccdB gene in vector Te pep.bbn is replaced by Temer00088,Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249,Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570,Temer08163 or Temer07305 expression cassettes according to routinecloning procedures. R. emersonii promoter 2, represented by SEQ ID NO:81, is cloned upstream of the R. emersonii Temer00088, Temer09484,Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848,Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163or Temer07305 coding region with A. nidulans amdS terminator, generatingconstruct pEBA. The A. nidulans amdS terminator sequence is representedby SEQ ID NO: 82. A schematic representation of pEBA for overexpressionof the Gene of interest (G01) being Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305is shown in FIG. 4.

Example 3: Overexpression of Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305Gene in Rasamsonia emersonii

Linear DNA of pEBA and pEBA1006 are isolated and used to transformRasamsonia emersonii using method as described earlier in WO2011/054899.The linear DNAs can integrate together into the genome at the RePepAlocus, thus substituting the RePepA gene by the Temer00088, Temer09484,Temer08028, Temer02362, Temer08862, Temer04790, Temer05249, Temer06848,Temer02056, Temer03124, Temer09491, Temer06400, Temer08570, Temer08163or Temer07305 and ble gene. Transformants are selected on phleomycinmedia and colony purified and tested according to procedures asdescribed in WO2011/054899. Growing colonies are diagnosed by PCR forintegration at the RePepA locus using a primer in the gpdA promoter ofthe deletion cassette and a primer directed against the genomic sequencedirectly upstream of the 5′ targeting region. Candidate transformants inwhich RePepA is replaced by Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 orTemer07305/ble cassettes are obtained.

Example 4: Enzymatic Activity in Temer00088, Temer09484, Temer08028,Temer02362, Temer08862, Temer04790, Temer05249, Temer06848, Temer02056,Temer03124, Temer09491, Temer06400, Temer08570, Temer08163 or Temer07305Overexpressing Rasamsonia emersonii Strains

Temer00088, Temer09484, Temer08028, Temer02362, Temer08862, Temer04790,Temer05249, Temer06848, Temer02056, Temer03124, Temer09491, Temer06400,Temer08570, Temer08163 or Temer07305 overexpressing strains arefermented in shake flask in Rasamsonia medium 3 and supernatants areanalysed for activity according to Table 1 in a suitable assay. Anincrease in activity is observed in supernatants of Temer00088,Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249,Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570,Temer08163 or Temer07305 overexpressing strains compared to thewild-type strain, indicating that overexpression of Temer00088,Temer09484, Temer08028, Temer02362, Temer08862, Temer04790, Temer05249,Temer06848, Temer02056, Temer03124, Temer09491, Temer06400, Temer08570,Temer08163 or Temer07305 improves activity in R. emersonii.

Example 5: Aspergillus niger Shake Flask Fermentation

About 10⁷ spores of selected transformants and control strains wereinoculated into 100 ml shake flasks with baffles containing 20 ml ofliquid pre-culture medium consisting of per liter: 30 g maltose.H₂O; 5 gyeast extract; 10 g hydrolyzed casein; 1 g KH₂PO₄; 0.5 g MgSO₄.7H₂O;0.03 g ZnCl₂; 0.02 g CaCl₂; 0.01 g MnSO₄.4H₂O; 0.3 g FeSO₄.7H₂O; 3 gTween 80; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml);pH5.5. These cultures were grown at 34 degrees Celsius for 16-24 hours.10 ml of this culture was inoculated into 500 ml shake flasks withbaffles containing 100 ml fermentation medium consisting of per liter:70 g glucose.H₂O; 25 g hydrolyzed casein; 12.5 g yeast extract; 1 gKH₂PO₄; 2 g K₂SO₄; 0.5 g MgSO₄.7H₂O; 0.03 g ZnCl₂; 0.02 g CaCl₂); 0.01 gMnSO₄.4H₂O; 0.3 g FeSO₄.7H₂O; 10 ml penicillin (5000 IU/ml)/Streptomycin(5000 UG/ml); adjusted to pH5.6. These cultures were grown at 34 degreesCelsius until all glucose was depleted (usually after 4-7 days). Samplestaken from the fermentation broth were centrifuged (10 min at 5000×g) ina swinging bucket centrifuge and supernatants collected and filteredover a 0.2 μm filter (Nalgene)

Shake Flask Concentration and Protein Concentration Determination withTCA-Biuret Method

In order to obtain greater amounts of material for further testing thefermentation supernatants obtained as described above (volume between 75and 100 ml) were concentrated using a 10 kDa spin filter to a volume ofapproximately 5 ml. Subsequently, the protein concentration in theconcentrated supernatant was determined via a TCA-biuret method.

Concentrated protein samples (supernatants) were diluted with water to aconcentration between 2 and 8 mg/ml. Bovine serum albumin (BSA)dilutions (0, 1, 2, 5, 8 and 10 mg/ml) were made and included as samplesto generate a calibration curve. Of each diluted protein sample 270 μlwas transferred into a 10 ml tube containing 830 μl of a 12% (w/v)trichloro acetic acid solution in acetone and mixed thoroughly.Subsequently, the tubes were incubated on ice water for one hour andcentrifuged for 30 minutes, at 4° C. and 6000 rpm. The supernatant wasdiscarded and pellets were dried by inverting the tubes on a tissue andletting them stand for 30 minutes at room temperature. Next, 3 mlBioQuant Biuret reagent mix was added to the pellet in the tube and thepellet was solubilized upon mixing followed by addition of 1 ml water.The tube was mixed thoroughly and incubated at room temperature for 30minutes. The absorption of the mixture was measured at 546 nm with awater sample used as a blank measurement and the protein concentrationwas calculated via the BSA calibration line.

Example 6: Identification of Thermophilic Rasamsonia emersoniiTemer09484 Beta-xylosidase Activity on Xylobiose

The beta-xylosidase activity of Rasamsonia emersonii Temer09484 wasanalysed as described above. The supernatant of the Temer09484 A. nigershake flask fermentation was concentrated and assayed in two dosages forxylose release from xylobiose after incubation for 24 hours at pH 4.5and 62° C. The enzyme showed significant xylose release from xylobioseas shown in Table 3. This shows that Temer09484 has beta-xylosidaseactivity.

TABLE 3 Effect of Rasamsonia emersonii Temer09484 on release of xylosefrom xylobiose (100 ug/mL) after 24 h incubation at pH 4.5 and 62° C.Protein ID Dosage (mg/g DM) Product xylose (ug/mL) No enzyme 0 0Temer09484 1 100 Temer09484 5 100

Example 7: Identification of Thermophilic Rasamsonia emersoniiTemer09484 Beta-xylosidase Activity on Polymeric Xylan Substrates

As a second experiment the activity of the beta-xylosidase activity ofRasamsonia emersonii Temer09484 was also analysed on polymeric xylansubstrates. The supernatant of the Temer09484 A. niger shake flask wasdosed at 10 mg/g to three different polymeric xylan substrates. From allthree substrates xylose was released (Table 4) while no xylooligomerswere formed. This shows that Temer09484 also has beta-xylosidaseactivity on polymeric substrates next to small oligomers as shown inExample 6.

TABLE 4 Effect of Rasamsonia emersonii Temer09484 on release of xylosefrom several xylan substrates after incubation for 24 h at pH 4.5 and60° C. at a dosage of 10 mg/g DM. ug/mL* Substrate (2 mg/mL) xyloseBeech wood xylan 320 Birch wood xylan 250 Oat arabinoxylan 334 * Allsubstrates contain < 3.0 ug/mL xylose when no enzyme was added

Example 8. Improvement of Two Different Cellulose Mixtures by Additionof Temer09484 for the Hydrolysis of Lignocellulosic Feedstocks

The supernatant of the Temer09484 A. niger shake flask fermentation wasconcentrated and spiked on a mild acid pretreated corn stover feedstockas described above. The enzyme showed significant xylose release fromthis feedstock in a wide range of temperatures (50, 65 and 75° C.) andpH values (3.5-4.5-5.0) used during the 72 hours of incubation as shownin Table 5. This shows that Temer09484 is important for the hydrolysisof lignocellulosic feedstocks.

TABLE 5 Effect of Rasamsonia emersonii Temer09484 on release of xylose(g/L) from mildly acid pretreated corn stover feedstock after 72 hincubation at different temperature/pH conditions. pH 5.0- pH 3.5- pH4.5- pH 4.5- Protein ID 50° C. 65° C. 65° C. 75° C. Feedstock only-noenzyme 0.14 0.14 0.14 0.13 Temer09484 0.29 0.22 0.23 0.19

The supernatant of the Temer09484 A. niger shake flask fermentation wasalso tested in combination with 2 different cellulose mixtures: TEC-210and Celluclast, both with additional BG added. The xylose release frommildly acid pretreated corn stover was improved for both cellulose mixesby the addition of Temer09484 in a wide range of temperatures (50, 65and 75° C.) and pH values (3.5-4.5-5.0) used during the 72 hours ofincubation as shown in Table 6. This shows that Temer09484 can be usedto improve cellulose mixes in a wide range of temperatures and pH valuesused for the hydrolysis of lignocellulosic feedstocks.

TABLE 6 Effect of Rasamsonia emersonii Temer09484 when spiked to twodifferent cellulose mixes on release of xylose (g/L) from mildly acidpretreated corn stover feedstock after 72 h incubation at differenttemperature/pH conditions. pH 5.0- pH 3.5- pH 4.5- pH 4.5- Protein ID50° C. 65° C. 65° C. 75° C. Feedstock only-no enzyme 0.14 0.14 0.14 0.13TEC-210 + 8% BG 0.51 0.45 0.57 0.22 TEC-210 + 8% BG + 0.63 0.53 0.630.33 Temer09484 Celluclast + 8% BG 0.49 0.23 0.27 0.19 Celluclast + 8%BG + 0.61 0.24 0.32 0.24 Temer09484

Example 9: Identification of Thermophilic Rasamsonia emersoniiTemer00088 Beta-xylosidase Activity on Xylobiose

The beta-xylosidase activity of Rasamsonia emersonii Temer00088 wasanalysed as described above. The supernatant of the Temer00088 A. nigershake flask fermentation was concentrated and assayed in two dosages forxylose release from xylobiose after incubation for 24 hours at pH 4.5and 62° C. The enzyme showed significant xylose release from xylobioseas shown in Table 7. This shows that Temer00088 has beta-xylosidaseactivity.

TABLE 7 Effect of Rasamsonia emersonii Temer00088 on release of xylosefrom xylobiose (100 ug/mL) after 24 h incubation at pH 4.5 and 62° C.Protein ID Dosage (mg/g DM) Product xylose (ug/mL) No enzyme 0 0Temer00088 1 76 Temer00088 5 99

Example 10: Identification of Thermophilic Rasamsonia emersoniiTemer00088 Beta-xylosidase Activity on Polymeric Xylan Substrates

As a second experiment the activity of the beta-xylosidase activity ofRasamsonia emersonii Temer00088 was also analysed on polymeric xylansubstrates. The supernatant of the Temer00088 A. niger shake flask wasdosed at 10 mg/g to three different polymeric xylan substrates. From allthree substrates xylose was released (Table 8) while no xylooligomerswere formed. This shows that Temer00088 also has beta-xylosidaseactivity on polymeric substrates next to small oligomers as shown inExample 9.

TABLE 8 Effect of Rasamsonia emersonii Temer00088 on release of xylosefrom several xylan substrates after incubation for 20 h at pH 4.5 and60° C. at a dosage of 10 mg/g DM. ug/mL* Substrate (2 mg/mL) xyloseBeech wood xylan 373 Birch wood xylan 469 Oat arabinoxylan 298 *Allsubstrates contain < 3.0 ug/mL xylose when no enzyme was added

Example 11. Improvement of Two Different Cellulose Mixtures by Additionof Temer00088 for the Hydrolysis of Lignocellulosic Feedstocks

The supernatant of the Temer00088 A. niger shake flask fermentation wasconcentrated and spiked on a mild acid pretreated corn stover feedstockas described above. The enzyme showed significant xylose release fromthis feedstock in a wide range of temperatures (50, 65 and 75° C.) andpH values (3.5-4.5-5.0) used during the 72 hours of incubation as shownin Table 3. This shows that Temer00088 is important for the hydrolysisof lignocellulosic feedstocks.

TABLE 9 Effect of Rasamsonia emersonii Temer00088 on release of xylose(g/L) from mildly acid pretreated corn stover feedstock after 72 hincubation at different temperature/pH conditions. pH 5.0- pH 3.5- pH4.5- pH 4.5- Protein ID 50° C. 65° C. 65° C. 75° C. Feedstock only-noenzyme 0.14 0.14 0.14 0.13 Temer00088 0.29 0.26 0.27 0.22

The supernatant of the Temer00088 A. niger shake flask fermentation wasalso tested in combination with 2 different cellulose mixtures: TEC-210and Celluclast, both with additional BG added. The xylose release frommildly acid pretreated corn stover was improved for both cellulose mixesby the addition of Temer00088 in a wide range of temperatures (50, 65and 75° C.) and pH values (3.5-4.5-5.0) used during the 72 hours ofincubation as shown in Table 10. This shows that Temer00088 can be usedto improve cellulose mixes in a wide range of temperatures and pH valuesused for the hydrolysis of lignocellulosic feedstocks.

TABLE 10 Effect of Rasamsonia emersonii Temer00088 when spiked to twodifferent cellulose mixes on release of xylose (g/L) from mildly acidpretreated corn stover feedstock after 72 h incubation at differenttemperature/pH conditions. pH 5.0- pH 3.5- pH 4.5- pH 4.5- Protein ID50° C. 65° C. 65° C. 75° C. Feedstock only-no enzyme 0.14 0.14 0.14 0.13TEC-210 + 8% BG 0.51 0.45 0.57 0.22 TEC-210 + 8% BG + Temer09484 0.620.59 0.67 0.39 Celluclast + 8% BG 0.49 0.23 0.27 0.19 Celluclast + 8%BG + Temer09484 0.66 0.29 0.35 0.27

Example 12: Identification of Thermophilic Rasamsonia emersoniiXyloglucan Specific Endoglucanase

The xyloglucanase activity of Rasamsonia emersonii Temer04790 wasanalysed as described above. The supernatant of the Temer04790 A. nigershake flask fermentation was concentrated, added to the substratexyloglucan and incubated for 24 hours at pH 4.5 and 60° C. The enzymewas able to release several oligomers as shown in FIG. 6. This showsthat Temer04790 is active on xyloglucan and releases similar oligomersas the commercial cellulase mix Celluclast from Trichoderma reesei.

To quantify the amount of oligomers formed reducing ends were measuredafter incubation of both Temer04790 and the cellulase mix.Carboxymethylcellulose was also used as substrate to determine thespecificity of the enzymes and a higher temperature, 75° C. was usednext to 60° C. Temer04790 is specific towards xyloglucan as hardly anyactivity on CMC was seen in contrast to the cellulase mixture (Table11). Furthermore, Temer04790 was still active at 75° C. while thecellulase mixture was almost inactive on xyloglucan at 75° C.

TABLE 11 Effect of Rasamsonia emersonii Temer04790 on the hydrolysis ofxyloglucan (tamarind) and carboxymethylcellulose (CMC) (Sigma) measuredby the formation of reducing ends expressed as glucose equivalents(ug/mL) after 24 h incubation at pH 4.5 at 60 ° C. and 75° C. 60° C. pH4.5 75° C. pH 4.5 xyloglucan CMC xyloglucan CMC no enzyme −17 −18 −18−18 Temer04790 92 8 69 −9 cellulase mix* 64 132 5 47 *Celluclast fromThrichoderma reesei (Sigma)

Example 13: Identification of Thermophilic Rasamsonia emersoniiArabinofuranosidase Activity

The arabinofuranosidase activity of Rasamsonia emersonii Temer05249 wasanalysed as described above. The supernatant of the Temer05249 A. nigershake flask fermentation was concentrated and added toarabinoxylooligomers at 10 mg/g followed by incubation for 24 hours atpH 4.5 and 65° C. The enzyme showed significant arabinose release fromarabinoxylooligomers as shown in Table 12. This shows that Temer05249has arabinofuranosidase activity.

TABLE 12 Effect of Rasamsonia emersonii Temer05249 on the release ofarabinose from wheat arabinoxylan, which was pre-incubated with anendo-xylanase, after incubation for 24 h at pH 4.5 and 65° C. at adosage of 10 mg/g DM. Protein ID Arabinose (ug/mL) No enzyme 4Temer05249 111

Example 14: Identification of Thermophilic Rasamsonia emersoniiEndo-xylanase Activity

The endo-xylanase activity of Rasamsonia emersonii Temer03124 wasanalysed as described above. The supernatant of the Temer03124 A. nigershake flask fermentation was concentrated and added to several xylansubstrates at 10 mg/g followed by incubation for 20 hours at pH 4.5 and60° C. The enzyme showed significant release of xylose and a range ofxylooligomers as shown in Table 13. This shows that Temer03124 hasendo-xylanase activity.

TABLE 13 Effect of Rasamsonia emersonii Temer03124 on release of xyloseand xylose oligomers from several xylan substrates after incubation for20 h at pH 4.5 and 60° C. at a dosage of 10 mg/g DM. ug/mL* Substrate (2mg/mL) xylose xylobiose xylotriose xylotetraose Beech wood xylan 34.312.6 10.4 11.4 Birch wood xylan 30.6 17.8 16.1 16.1 Oat arabinoxylan27.3 17.2 12.5 10.9 Wheat arabinoxylan 33.4 36.9 15.0 5.2 *Allsubstrates contain <3.5 ug/mL of each product measured if no enzyme isadded

Example 15: Identification of Thermophilic Rasamsonia emersoniiEndo-xylanase Activity

The endo-xylanase activity of Rasamsonia emersonii Temer08570 wasanalysed as described above. The supernatant of the Temer08570 A. nigershake flask fermentation was concentrated and added to several xylansubstrates at 10 mg/g followed by incubation for 20 hours at pH 4.5 and60° C. The enzyme showed significant release of xylose and a range ofxylooligomers as shown in Table 14. This shows that Temer08570 hasendo-xylanase with xylobiose, xylotriose and xylotetraose as mainproducts.

TABLE 14 Effect of Rasamsonia emersonii Terner08570 on release of xyloseand xylose oligomers from several xylan substrates after incubation for20 h at pH 4.5 and 60° C. at a dosage of 10 mg/g DM. ug/mL* Substrate (2mg/mL) xylose xylobiose xylotriose xylotetraose Beech wood xylan 5 19 2525 Birch wood xylan 4 14 17 17 Oat arabinoxylan 0 16 18 15 *Allsubstrates contain <3.5 ug/mL of each product measured if no enzyme isadded

Example 16: Identification of Thermophilic Rasamsonia emersoniiEndo-xylanase Activity

The endo-xylanase activity of Rasamsonia emersonii Temer08163 wasanalysed as described above. The supernatant of the Temer08163 A. nigershake flask fermentation was concentrated and added to several xylansubstrates at 10 mg/g followed by incubation for 20 hours at pH 4.5 and60° C. The enzyme showed significant release of xylbiose and xylose asshown in Table 15. This shows that Temer08570 has endo-xylanase activitywith xylobiose as main product which was 12-25 times higher than theamount of xylose released.

TABLE 15 Effect of Rasamsonia emersonii Temer08163 on release of xyloseand xylose oligomers from several xylan substrates after incubation for20 h at pH 4.5 and 60° C. at a dosage of 10 mg/g DM. ug/mL* Substrate (2mg/mL) xylose xylobiose xylotriose xylotetraose Beech wood xylan 22.4581.4 0 0 Birch wood xylan 30.9 527.3 0 0 Oat arabinoxylan 13.6 205.1 00 Wheat arabinoxylan 5.4 65.9 0 0 *All substrates contain <3.5 ug/mL ofeach product measured.

Example 17: Identification of Thermophilic Rasamsonia emersoniiAlpha-glucuronidase Activity

The alpha-glucuronidase activity of Rasamsonia emersonii Temer07305 wasanalysed as described above. The supernatant of the Temer07305 A. nigershake flask fermentation was concentrated and added to aldouronic acidsboth 1 and 10 mg/g followed by incubation for 24 hours at pH 4.5 and 60°C. The enzyme was able to remove 4-O-methylglucuronic acid from thexyloilogomers resulting in the simultaneous release of xylose,xylobiose, xylotriose and xylotatraose as shown in Table 16. This showsthat Temer07305 has alpha-glucuronidase activity.

TABLE 16 The release of xylose and xylose oligomers by Rasamsoniaemersonii Temer07305 from aldouronic acids as a result of the hydrolysisof 4-O-methylglucuronic acid from these xylooligomers, after incubationfor 24 h at pH 4.5 and 60° C. at a dosage of 1 and 10 mg/g DM. Dosage(mg/g Area/(mg/mL) substrate Protein ID DM) xylose xylobiose xylotriosexylotetraose No enzyme x 25 6 0 0 Temer07305 1 45 180 58 19 Temer0730510 120 180 55 9

1. A polypeptide having hemicellulase activity, comprising: (i) an aminoacid sequence having at least 85% sequence identity to SEQ ID NO:72 andcontaining at least one substitution modification relative to saidsequence; or (ii) an amino acid sequence containing at least onesubstitution modification relative to SEQ ID NO:72, encoded by a nucleicacid sequence having at least 85% sequence identity to a sequenceselected from the group consisting of SEQ ID NOs: 71, 74, and
 75. 2. Thepolypeptide according to claim 1, said polypeptide havingalpha-glucuronidase activity.
 3. A nucleic acid sequence coding for ahemicellulase, whereby the nucleic acid sequence is selected from thegroup consisting of: (a) a nucleic acid sequence having at least 85%sequence identity with a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 71, 74, and 75, and containing at least onesubstitution modification relative to said nucleic acid sequence; (b) anucleic acid sequence hybridizing with the complement of a nucleic acidsequence having at least 85% sequence identity with a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 71, 74, and75, and containing at least one substitution modification relative tosaid nucleic acid sequence; (c) a nucleic acid sequence encoding anamino acid sequence having at least 85% sequence identity with SEQ IDNO:72, and containing at least one substitution modification relative tosaid sequence; or (d) a nucleic acid sequence which is the reversecomplement of a nucleic acid sequence as defined in (a), (b) or (c). 4.A nucleic acid construct or vector comprising the nucleic acid sequenceaccording to claim
 3. 5. A cell comprising the polypeptide according toclaim 1, optionally the cell is a fungal cell, optionally a fungal cellselected from the group consisting of the genera Acremonium, Agaricus,Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia,Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
 6. The cellaccording to claim 5, wherein one or more gene is deleted, knocked-outor disrupted in full or in part, wherein optionally the gene encodes fora protease.
 7. A method for preparing the polypeptide according to claim1, having hemicellulase activity, which method comprises cultivating acell under conditions which allow for expression of said polypeptideand, optionally, recovering the expressed polypeptide.
 8. A compositioncomprising: (i) the polypeptide according to claim 1 and; (ii) acellulase and/or an additional hemicellulase and/or a pectinase.
 9. Thecomposition according to claim 8, wherein the cellulase is a GH61,cellobiohydrolase, cellobiohydrolase I, cellobiohydrolase II,endo-β-1,4-glucanase, β-glucosidase or β-(1,3)(1,4)-glucanase.
 10. Thecomposition according to claim 8, wherein the additional hemicellulaseis an endoxylanase, β-xylosidase, α-L-arabinofuranosidase,α-D-glucuronidase, cellobiohydrolase, feruloyl esterase, coumaroylesterase, α-galactosidase, β-galactosidase, β-mannanase orβ-mannosidase.
 11. The polypeptide of claim 1, comprising: (i) an aminoacid sequence having at least 90% sequence identity to SEQ ID NO:72, andcontaining at least one substitution modification relative to saidsequence; or (ii) an amino acid sequence containing at least onesubstitution modification relative to SEQ ID NO:72, encoded by a nucleicacid sequence having at least 90% sequence identity to a sequenceselected from the group consisting of SEQ ID NOs: 71, 74, and
 75. 12.The polypeptide of claim 1, comprising: (i) an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:72, and containing atleast one substitution modification relative to said sequence; or (ii)an amino acid sequence containing at least one substitution modificationrelative to SEQ ID NO:72, encoded by a nucleic acid sequence having atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 71, 74, and
 75. 13. A method for treating asubstrate comprising hemicellulose, optionally a plant material, whichmethod comprises contacting the substrate with the polypeptide accordingto claim
 1. 14. A method for producing a sugar from a lignocellulosicmaterial, which method comprises contacting the lignocellulosic materialwith the polypeptide according to claim
 1. 15. A method for producing asugar from a lignocellulosic material, which method comprises contactingthe composition according to claim 8 with the lignocellulosic material.16. A method for treating a substrate comprising hemicellulose,optionally a plant material, which method comprises contacting thesubstrate with the composition according to claim
 8. 17. A method forthe preparation of a fermentation product, which method comprises: (a)performing the method according to claim 13; and (b) fermentation of theresulting material, to thereby prepare the fermentation product.
 18. Amethod for the preparation of a fermentation product, which methodcomprises: (a) performing the method according to claim 16; and (b)fermentation of the resulting material, to thereby prepare thefermentation product.
 19. The method of claim 13, wherein said substratecomprises fiber from corn kernels.
 20. The method of claim 16, whereinsaid substrate comprises fiber from corn kernels.
 21. The method ofclaim 14, wherein the lignocellulosic material comprises fiber from cornkernels.
 22. The method of claim 15, wherein the lignocellulosicmaterial comprises fiber from corn kernels.
 23. A method for producing asugar from a lignocellulosic material, comprising: (a) pretreatment ofthe lignocellulosic material; and (b) contacting the pretreatedlignocellulosic material with a polypeptide having hemicellulaseactivity, comprising: (i) an amino acid sequence having at least 85%sequence identity to SEQ ID NO:72; (ii) an amino acid sequence encodedby a nucleic acid sequence having at least 85% sequence identity to asequence selected from the group consisting of SEQ ID NOs: 71, 74, and75; (iii) an amino acid sequence encoded by a nucleic acid sequencehybridizing with the complement of a nucleic acid sequence having atleast 85% sequence identity with a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 71, 74, and 75; or (iv) an aminoacid sequence encoded by a nucleic acid sequence which is the reversecomplement of a nucleic acid sequence as defined in (i), (ii) or (iii).24. The method of claim 23, further comprising contacting thelignocellulosic material with a cellulase and/or an additionalhemicellulase and/or a pectinase.
 25. The method of claim 24, whereinthe cellulase is selected from the group consisting of a GH61,cellobiohydrolase, cellobiohydrolase I, cellobiohydrolase II,endo-β-1,4-glucanase, β-glucosidase, and β-(1,3)(1,4)-glucanase.
 26. Themethod of claim 24, wherein the additional hemicellulase is selectedfrom the group consisting of endoxylanase, β-xylosidase,α-L-arabinofuranosidase, α-D-glucuronidase, cellobiohydrolase, feruloylesterase, coumaroyl esterase, α-galactosidase, β-galactosidase,β-mannanase, and β-mannosidase.
 27. The method of claim 24, wherein thepectinase is selected from the group consisting ofendo-polygalacturonase, pectin methyl esterase, endo-galactanase,β-galactosidase, pectin acetyl esterase, endo-pectin lyase, pectatelyase, α-rhamnosidase, exo-galacturonase, exo-polygalacturonate lyase,rhamnogalacturonan hydrolase, rhamnogalacturonan lyase,rhamnogalacturonan acetyl esterase, rhamnogalacturonangalacturonohydrolase, xylogalacturonase, and α-arabinofuranosidase. 28.The method of claim 23, wherein the lignocellulosic material comprisesfiber from corn kernels.
 29. A method for preparing a fermentationproduct, comprising: (a) performing the method according to claim 23;and (b) fermentation of the resulting material, to thereby prepare thefermentation product.
 30. The method of claim 23, wherein saidpolypeptide has alpha-glucuronidase activity.
 31. A method for treatinga substrate comprising hemicellulose, comprising: (a) pretreatment ofthe substrate; and (b) contacting the pretreated substrate with apolypeptide having hemicellulase activity, comprising: (i) an amino acidsequence having at least 85% sequence identity to SEQ ID NO:72; (ii) anamino acid sequence encoded by a nucleic acid sequence having at least85% sequence identity to a sequence selected from the group consistingof SEQ ID NOs: 71, 74, and 75; (iii) an amino acid sequence encoded by anucleic acid sequence hybridizing with the complement of a nucleic acidsequence having at least 90% sequence identity with a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 71, 74, and75; or (iv) an amino acid sequence encoded by a nucleic acid sequencewhich is the reverse complement of a nucleic acid sequence as defined in(i), (ii) or (iii).
 32. The method of claim 31, further comprisingcontacting the lignocellulosic material with a cellulase and/or anadditional hemicellulase and/or a pectinase.
 33. The method of claim 32,wherein the cellulase is selected from the group consisting of a GH61,cellobiohydrolase, cellobiohydrolase I, cellobiohydrolase II,endo-β-1,4-glucanase, β-glucosidase, and β-(1,3)(1,4)-glucanase.
 34. Themethod of claim 32, wherein the additional hemicellulase is selectedfrom the group consisting of endoxylanase, β-xylosidase,α-L-arabinofuranosidase, α-D-glucuronidase, cellobiohydrolase, feruloylesterase, coumaroyl esterase, α-galactosidase, β-galactosidase,β-mannanase, and β-mannosidase.
 35. The method of claim 32, wherein thepectinase is selected from the group consisting ofendo-polygalacturonase, pectin methyl esterase, endo-galactanase,β-galactosidase, pectin acetyl esterase, endo-pectin lyase, pectatelyase, α-rhamnosidase, exo-galacturonase, exo-polygalacturonate lyase,rhamnogalacturonan hydrolase, rhamnogalacturonan lyase,rhamnogalacturonan acetyl esterase, rhamnogalacturonangalacturonohydrolase, xylogalacturonase, and α-arabinofuranosidase. 36.The method of claim 31, wherein said substrate comprises a plantmaterial.
 37. The method of claim 31, wherein said substrate comprisesfiber from corn kernels.
 38. A method for the preparation of afermentation product, which method comprises: (a) performing the methodaccording to claim 31; and (b) fermentation of the resulting material,to thereby prepare the fermentation product.
 39. A method for preparinga polypeptide having hemicellulase activity, comprising cultivating acell under conditions which allow for expression of said polypeptideand, optionally, recovering the expressed polypeptide, wherein saidpolypeptide comprises: (i) an amino acid sequence having at least 85%sequence identity to SEQ ID NO:72; (ii) an amino acid sequence encodedby a nucleic acid sequence having at least 85% sequence identity to asequence selected from the group consisting of SEQ ID NOs: 71, 74, and75; (iii) an amino acid sequence encoded by a nucleic acid sequencehybridizing with the complement of a nucleic acid sequence having atleast 85% sequence identity with a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 71, 74, and 75; or (iv) an aminoacid sequence encoded by a nucleic acid sequence which is the reversecomplement of a nucleic acid sequence as defined in (i), (ii) or (iii).